In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2p are presented. These are the first atomic structures of the cubic core and of the E3 component of the pyruvate dehydrogenase and related multienzyme complexes. The structural features of the two enzymes are analyzed in the context of their catalytic functioning and of their participation to the overall activity of the oxoacid dehydrogenases. ...

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Mattevi, Andrea

English

Dissertations of the University of Groningen

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

Structure of the cubic core and the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex.

Proceedings - University of Groningen

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

Mattevi, Andrea,

University of Groningen Digital Archive

University of Groningen Research Database

  
 University of Groningen
Structure of the cubic core and the lipoamide dehydrogenase component of the pyruvate
dehydrogenase complex.
Mattevi, Andrea
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1992
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of the
pyruvate dehydrogenase complex. s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
SUMMARY
The pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidative
decarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of a
molecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs at
the junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoacid
dehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-
oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).
These complexes are large systems having a molecular weight of 5-10 million daltons and
comprising multiple copies of no less than three different enzymes. The decarboxylase
component (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylation
of the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 then
transfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter is
reoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers the
reducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)
and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH are
oxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH has
its own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all these
multienzyme systems.
In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2
carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysine
side chain. Moreover, EZ forms the inner core of the complexes to which the peripheral
members El and E3 are bound. Consistent with this essential roLe,E2 has a unique modular
anangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residues
long, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50
residues, is essential for the association of the peripheral subunits; and, (iii) the catalytic
domain of approximately 250 residues, possesses the active site and forms the inner higtÍy
symmetrical'r'n core of the complexes. The domains are connected to each other by higtrly
flexible polypeptide regions, which Íue essential for the functioning of the systems. Thanks
to the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactor
to visit the catalytic centers of the EL,E2 and E3 enzymes.
In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2p
are presented. These aÍe the first atomic structures of the cubic core and of the E3 component
of the pyruvate dehydrogenase and related multienzyme complexes. The structural features
of the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of their
participation to the overall activity of the oxoacid dehydrogenases.
Lipoamide dehydrogenase (E3)
The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solved
by Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The
180
results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme are
presented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2
Á resolution. Each monomer of the E3 dimer comprises four domains and the active site is
located at the interface between the two subunits. The unique combination of the flavin ring
with an adjacent disulphide bridge generates the redox center involved in the electron transfer
reaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ and
the lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.
The overall sffucture of the E3 appeaÍs to be similar to that of human glutathione
reductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparison
between the two enzymes reveals a number of differences in their tertiary and quatemary
structure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the
interface domain appear to be differently oriented with respect to the FAD-binding domain
by 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respect
to each other by more than 4 À, when their mutual position is compared with that observed
in glutathione reductase. Remarkably, in spite of these modifications the catalytic centers
remain virtually identical in the two enzymes.
The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*
is presented in chapter 3. The structure was solved by molecular replacement and refined at
2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putida
E3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrate
occupies an analogous position as in the structure of glutathione reductase, whereas the
nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine
ring and points into solution. This suggests a dependence of the substrate binding on the
redox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformation
of the C-terminal residues which fold 'back' into the putative lipoamide binding region. The
C-terminus has been proven to be important for activity by site directed mutagenesis.
However, the distance of the C-terminus to the catalytically essential residues is still
surprisingly large, over 6 À, and the precise role of the C-terminus still needs to be
elucidated.
Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to the
same protein from A. vinelandii. However, the two enzymes present significant differences
in their thermostability and redox properties. The melting temperature of P.fluorescensE3
is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescens
E3 cannot be reduced to the EHo state, in which both the flavin ring and the catalytic
disulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues about
the causes of these differences between the two proteins, the structure of P. fluorescens E3
was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.
As expected, the structure of P. fluorescens E3 is closely related to that of the same
flavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Ca
atoms. The residues in the active site are identical. while 89Vo of. the interface residues are
181
the same in the two proteins. Thus, only few structural variations aÍe responsible for the
differences in thermostability and redox properties between the two homologous proteins.
Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leaves
a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,
unpaired to any H-bond donor. This might account for the greater stability of P. fluorescens
E3 with respect to the A. vínelandii enzyme.
The catalytic domain of dihydrolipoyl transacetylase (E2p)
The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvate
dehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.
As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multiple
isomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 A
resolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of
120 À and pores of a diameter of 30 À crossing each face of the cube. The true building
block is the E2p trimer, eight of which occupy the comers of the cube. The trimer is
stabilized by extensive intermolecular interactions between threefold related subunits, whereas
the interactions along the twofold axis leading to the assembly of the trimers into the cubic
24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. The
topology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase
(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identity
between the two enzymes is only I7Vo. A remarkable difference between E2pCD and CAT
is given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminal
remainder of the subunit, while in E2pCD they form an aÍrn which interacts with a
neighboring subunit, thus lacing the subunits of the trimer, together.
The catalytic center of E2pCD is located at the interface between two threefold related
subunits and consists of a 30 À long channel, whose two entraÍrces form the CoA and
lipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA must
enter its binding site from the inside of the cube, whereas lipoamide enters from the outside.
In this way, the flexible lipoyl domains on the surface of the cubic core will allow the
covalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits El
and E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on the
crystal structures of E2pCD and E3, combined to the wealth of electron microscopy and
mutagenesis data.
The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where the
structure of six complexes and derivatives of A. vinelandii EZpCD are described. The binary
complexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,
respectively.The two substrates Íue bound in an extended conformation, at the two opposite
entrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates are
in hydrogen-bond distance from the side chain of His 610, supporting the indication, derived
from the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts
182
1
i
as general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonation
is the first step of the reaction catalyzed by E2pCD, which is thought to proceed via a
negatively charged tetrahedral intermediate. The binary complex between E2pCD and
hydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle of
the catalytic center, which is therefore capable of hosting and stabilizing a negative charge.
The structure of the binary complex with hydrogen sulphite further suggests that transition
state stabilization can be provided by a direct hydrogen-bond between the side chain of Ser
558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of a
ternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals that
CoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoA
adopts a helical conformation with two intramolecular hydrogen bonds and the reactive
sulphur of the pantetheine aÍm positioned 12.A away from the active site residues involved
in the transferase reaction.
The structures of the binary and temary complexes between E2pCD and its substrates offer
a clue for understanding the different substrate specificities of the various E2 transacylases
forming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of the
three-dimensional structure of E2pCD combined with the analysis of the amino acid
sequences suggests that the natuÍe and size of only a few side chains part of the catalytic
center might be the major determinants for tuning the substrate specificity among the various
E2 transacylases.
1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.
2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.
3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.
Biochem., 20I, 203-209.
4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),
2, L-48, Academic Press, London.
5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.
6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.
7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).
183


University of Groningen

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

ARTS repository - University of Groningen

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

NARCIS 

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

   University of GroningenStructure of the cubic core and the lipoamide dehydrogenase component of the pyruvatedehydrogenase complex.Mattevi, AndreaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1992Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mattevi, A. (1992). Structure of the cubic core and the lipoamide dehydrogenase component of thepyruvate dehydrogenase complex. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022SUMMARYThe pyruvate dehydrogenase multienzyme complex (PDH)*rt catalyzes the oxidativedecarboxylation of pyÍuvate coupled to the synthesis of acetylCoA and to the reduction of amolecule of NAD*. This is a key reaction in the metabolism of glucose, because it occurs atthe junction of glycolysis and the Krebs cycle. PDH is a member of the family of the oxoaciddehydrogenase multienzyme complexes, which, in addition to PDH, also comprises the 2-oxoglutarate dehydrogenase (OGDH) and the branched-chain oxoacid dehydrogenase (BCDH).These complexes are large systems having a molecular weight of 5-10 million daltons andcomprising multiple copies of no less than three different enzymes. The decarboxylasecomponent (El), a thiamin pyrophosphate dependent protein, ca:ries out the decarboxylationof the oxoacid and attaches the acyl group to lipoamide. The transacylase enzyme E2 thentransfers the acyl moiety to CoA, releasing lipoamide in the reduced state. The latter isreoxidized by the FAD dependent enzyme lipoamide dehydrogenase (E3), which transfers thereducing equivalents to NAD". Specific components of PDH aÍe pyruvate decarboxylase (Elp)and dihydrolipoyl transacetylase (E2p), whereas the corresponding enzymes of OGDH areoxoglutarate dehydrogenase (Elo) and dihydrolipoyl transsuccinylase (E2o). Also BCDH hasits own specific Elb and E2b enzymes. Thus, E3 is the only component cornnon to all thesemultienzyme systems.In all oxoacid dehydrogenases E2 plays a key functional and structural role. Namely, E2carries the lipoamide cofactor, which is covalently bound to the e-amino group of a lysineside chain. Moreover, EZ forms the inner core of the complexes to which the peripheralmembers El and E3 are bound. Consistent with this essential roLe,E2 has a unique modularanangement'r'2'3, comprising thÍee types of domains: (i) the lipoyl domain about 100 residueslong, bears the covalently bound lipoamide; (ii) the EUgl binding domain comprising 50residues, is essential for the association of the peripheral subunits; and, (iii) the catalyticdomain of approximately 250 residues, possesses the active site and forms the inner higtÍysymmetrical'r'n core of the complexes. The domains are connected to each other by higtrlyflexible polypeptide regions, which Íue essential for the functioning of the systems. Thanksto the linkers, the lipoyl domains form a "super-arm", which allows the lipoamide cofactorto visit the catalytic centers of the EL,E2 and E3 enzymes.In this thesis, the crystal structures of the E3 component and of the catalytic domain of E2pare presented. These aÍe the first atomic structures of the cubic core and of the E3 componentof the pyruvate dehydrogenase and related multienzyme complexes. The structural featuresof the tv/o enzymes aÍe analyzed in the context of their catalytic functioning and of theirparticipation to the overall activity of the oxoacid dehydrogenases.Lipoamide dehydrogenase (E3)The crystal structure of Azotobacter vinelandii lipoanride dehydrogenase was initially solvedby Schierbeek et al.*rs using a combination of isomorphous and molecular replacement. The180results of the crystallographic refinement of this 100,000 daltons dimeric flavoenzyme arepresented in chapter 2.T\e final model has an R-factor of 19.\Vo for 44,292 reflections at2.2Á resolution. Each monomer of the E3 dimer comprises four domains and the active site islocated at the interface between the two subunits. The unique combination of the flavin ringwith an adjacent disulphide bridge generates the redox center involved in the electron transferreaction between the substrates,lipoamide and NAD*. The flavin ring separates the NAD+ andthe lipoamide binding sites, positioned on the two opposite faces of the reactive ring of FAD.The overall sffucture of the E3 appeaÍs to be similar to that of human glutathionereductase*t'u, h agreement with the 26Vo sequence identity. Nevertheless, a comparisonbetween the two enzymes reveals a number of differences in their tertiary and quatemarystructure. In each subunit of lipoamide dehydrogenase the NAD-binding domain and theinterface domain appear to be differently oriented with respect to the FAD-binding domainby 7.1" and 7.8", respectively. Furthermore, the two subunits of E3 are shifted with respectto each other by more than 4 À, when their mutual position is compared with that observedin glutathione reductase. Remarkably, in spite of these modifications the catalytic centersremain virtually identical in the two enzymes.The crystal structure of the complex between Pseudomonas putída E3 and the substrate NAD*is presented in chapter 3. The structure was solved by molecular replacement and refined at2.45 A resolution to a final crystallographic R-factor of 2I.3Vo.In this crystal form P. putidaE3 contains one NAD* molecule per subunit. The adenine-ribose moiety of the substrateoccupies an analogous position as in the structure of glutathione reductase, whereas thenicotinamide-ribose moiety is far removed from its expected position near the isoalloxazinering and points into solution. This suggests a dependence of the substrate binding on theredox state of the protein. Furthermore, the structure of P. putida E3 reveals the conformationof the C-terminal residues which fold 'back' into the putative lipoamide binding region. TheC-terminus has been proven to be important for activity by site directed mutagenesis.However, the distance of the C-terminus to the catalytically essential residues is stillsurprisingly large, over 6 À, and the precise role of the C-terminus still needs to beelucidated.Pseudomonas fluorescens lipoamide dehydrogenase has a high 84Vo seqtence identity to thesame protein from A. vinelandii. However, the two enzymes present significant differencesin their thermostability and redox properties. The melting temperature of P.fluorescensE3is 6o higher than that of the A. vinelandii enzyme. Moreover, unlike the latter, P. fluorescensE3 cannot be reduced to the EHo state, in which both the flavin ring and the catalyticdisulphide are completely reduced (FADHT.SH.SH)*i7. In order to obtain some clues aboutthe causes of these differences between the two proteins, the structure of P. fluorescens E3was solved by molecular replacement and refined to an R-factor of L9.4Vo at2.8 À resolution.As expected, the structure of P. fluorescens E3 is closely related to that of the sameflavoprotein from Azotobacter vinelandií with an r.m.s. difference of 0.53 À for 932 Caatoms. The residues in the active site are identical. while 89Vo of. the interface residues are181the same in the two proteins. Thus, only few structural variations aÍe responsible for thedifferences in thermostability and redox properties between the two homologous proteins.Among them, in the A. vinelandii molecule a threonine to alanine (T4524) mutation leavesa buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring,unpaired to any H-bond donor. This might account for the greater stability of P. fluorescensE3 with respect to the A. vínelandii enzyme.The catalytic domain of dihydrolipoyl transacetylase (E2p)The catalytic domain of A. vinelandíi E2p (residues 386-637) forms the core of the pyruvatedehydrogenase, by assembling 24 subunits into oligomers, having octahedral 432 symmetry.As described in chapters 5 and 6, the structure of A. vinelandiiEZpCD was solved by multipleisomorphous replacement and refined to a crystallographic R-factor of LïJVo at 2.6 Aresolution. The 24 subunits appeaÍ to be arranged in a hollow cubic structure with edges of120 À and pores of a diameter of 30 À crossing each face of the cube. The true buildingblock is the E2p trimer, eight of which occupy the comers of the cube. The trimer isstabilized by extensive intermolecular interactions between threefold related subunits, whereasthe interactions along the twofold axis leading to the assembly of the trimers into the cubic24-mer involve only a few C-terminal residues, with four inter-subunit hydrogen bonds. Thetopology of each catalytic domain subunit is similar to chloramphenicol acetyltransferase(CAT), comprising a central p sheet surrounded by five cr helices. The sequence identitybetween the two enzymes is only I7Vo. A remarkable difference between E2pCD and CATis given by the N-terminal residues. In CAT, these fold back onto the carboxyl-terminalremainder of the subunit, while in E2pCD they form an aÍrn which interacts with aneighboring subunit, thus lacing the subunits of the trimer, together.The catalytic center of E2pCD is located at the interface between two threefold relatedsubunits and consists of a 30 À long channel, whose two entraÍrces form the CoA andlipoamide binding sites. In the 24-meric cube, the trimers are oriented such that CoA mustenter its binding site from the inside of the cube, whereas lipoamide enters from the outside.In this way, the flexible lipoyl domains on the surface of the cubic core will allow thecovalently bound lipoamide to visit the active sites of E2 and of the peripheral subunits Eland E3. In chapter 5, a model for the entiÍe cubic-octahedral PDH is presented, based on thecrystal structures of E2pCD and E3, combined to the wealth of electron microscopy andmutagenesis data.The substrate binding and the catalytic functioning in E2p is analyzed in chapter 7, where thestructure of six complexes and derivatives of A. vinelandii EZpCD are described. The binarycomplexes of the enzyme with CoA and lipoamide were determined at 2.6 À and 3.0 À,respectively.The two substrates Íue bound in an extended conformation, at the two oppositeentrances of the 30 À long catalytic channel. The reactive thiol groups of both substrates arein hydrogen-bond distance from the side chain of His 610, supporting the indication, derivedfrom the similarity with chloramphenicol acetyl transferase, that this histidine side chain acts1821ias general base catalyst in the deprotonation of the reactive thiol of CoA. The deprotonationis the first step of the reaction catalyzed by E2pCD, which is thought to proceed via anegatively charged tetrahedral intermediate. The binary complex between E2pCD andhydrogen sulphite supports this hypothesis, showing that the anion is bound in the middle ofthe catalytic center, which is therefore capable of hosting and stabilizing a negative charge.The structure of the binary complex with hydrogen sulphite further suggests that transitionstate stabilization can be provided by a direct hydrogen-bond between the side chain of Ser558 and the oxyanion of the putative intermediate. Quite unexpectedly, the structure of aternary complex in which CoA and Lip(SH), are simultaneously bound to E2pCD, reveals thatCoA has an altemative, non-productive, binding mode. In the abortive temary complex, CoAadopts a helical conformation with two intramolecular hydrogen bonds and the reactivesulphur of the pantetheine aÍm positioned 12.A away from the active site residues involvedin the transferase reaction.The structures of the binary and temary complexes between E2pCD and its substrates offera clue for understanding the different substrate specificities of the various E2 transacylasesforming the core of oxoacid dehydrogenase multienzyme complexes. The investigation of thethree-dimensional structure of E2pCD combined with the analysis of the amino acidsequences suggests that the natuÍe and size of only a few side chains part of the catalyticcenter might be the major determinants for tuning the substrate specificity among the variousE2 transacylases.1. Reed, L.J. (1974). Acc. Chem. Res., Z, 40-46.2. Perham, R.N. (1991) Biochemistry, 30, 8501-8512.3. Robien A. et aL. (1992). Biochemistry, 3r, 3463-347r; Dardel F. et al. (1991), Eur. J.Biochem., 20I, 203-209.4. Oliver, R.M. & Reed, L.J. (1982). In "Electron microscopy of proteins" (Ha:ris, J.R. ed.),2, L-48, Academic Press, London.5. Schierbeek A.J. et al. (1989), J. Mol. Biol., 206,365-379.6. Karplus, P.A. & Schulz, G.E. (1987), J. Mol. Biol., I95,701-729.7. Benen, J.A.E. (1992). Ph.D. Thesis, University of Wageningen (The Netherlands).183

Structure of the cubic core and the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex.

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