Oubri, Arthur

English

University of Groningen

University of GroningenStructure and mechanism of soluble quinoprotein glucose dehydrogenaseOubri, ArthurIMPORTANT 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:1999Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Oubri, A. (1999). Structure and mechanism of soluble quinoprotein glucose dehydrogenase. [Thesis fullyinternal (DIV), University of Groningen]. [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: 30-12-2025SummarvDehydrogenases and oxidases participate in a variety of metabolic pathways. Amonst others theyconvert alcohols into carbonyl compounds. Three distinct classes of such proteins use threedifferent types of organic cofactors: nicotinamides, flavins, and quinone-containing compounds.The three-dimensional structures and catalytic mechanisms of the nicotinamide- and flavin-dependent enzymes have been characterized well. Both classes employ a catalytic mechanism thatinvolves the direct transfer of a hydride anion from the substrate to the cofactor. In contrast, muchless is known about how the quinone cofactor-containing enzymes perform catalysis.This thesis describes the elucidation of the three-dimensional structure and catalyticmechanism of a soluble glucose dehydrogenase (s-GDH) by X-ray crystallography. This enzymeuses pyrroloquinoline quinone (PQQ) as a cofactor to oxidize glucose and a variety of other sugarsto their corresponding lactones. In Chapter I the current knowledge of the chemistry of PQQ andPQQ-dependent proteins is reviewed. Furthermore, several possible reaction mechanisms arediscussed, which differ in the identity of the reactive moiety of PQQ (C4 or C5 carbonyl group)and in the nature of the intermediate (substrate or hydride addition to the cofactor).The crystallization, structure determination and refinement of the s-GDH structure aÍedescribed in Chapter 2. The three-dimensional structure of s-GDH was determined using themultiple isomorphous replacement method. The initial experimental phases were improved by acombination of solvent flattening and non-crystallographic symmetry averaging, resulting in aninterpretable electron density map. A nearly complete model was built and refined at 1.7 Àresolution.s-GDH functions as a homodimer. Each subunit has a p-propeller fold, formed by six four-stranded anti-parallel p-sheets, aligned around a six-fold pseudosymmetry axis. One monomerbinds three calcium ions. Two are located in the dimer interface, whereas the third is bound in theactive site. The discovery of four uncharacterized protein sequences that are homologous to s-GDHare also described in Chapter 2. These homologs may have a similar fold and catalyze similarPQQ-dependent reactions. A structure based sequence alignment of the six p-sheets in the s-GDHstructure reveals a six times repeated segment, which gives rise to a number of polar interactionswith a stabilizine function.Sunttt tu rtThe high resolution structurc of s-GDH in complex with the cofactor PQQ and thecompetlt ive inhibitor methylhydrazine is presented in Chapter 3. I t  affords a detai led view of theactive site with bound cofactor. PQQ binds to the active site calcium in a largc solvent accessibleclcÍi in the centre of each rnonomer. The binding of the cofactor is rnainly governed by polarinteractions. Methylhydrazine is covalently bound to the C5 atom of PQQ, which is confirrnecl to bethe rnost reactive rnoiety towards nucleophil ic compounds. Polarization of the C5-O5 bond by theside chain of Arg228 and the calcium ion may enhance the reactivi ty of the C5 atom. This indicatesthat the catalyt ic mechanisrn most l ikely involves nucleophil ic attack on the C5 atom, thus resolvingone of the two controversies in PQQ-assisted enzymatic catalysis.In Chapter 4 a complex consist ing of s-GDH, reduced PQQ and substrate is described.Glucose docks onto the surl'ace of PQQ and makes hydrogen bonds with several protein sidechains. In combination with biochemical and kinetic data, the arrangement of PQQ, calciurn,glucttse and Hisl,14 provicles conclusive evidence Íbr the catalyt ic mechanism: The side chain ofHisl4zl abstracts the proton Í iom the Ol hydroxyl group. This occurs in a fast step together withthe transfcr of a hydride from the substrate to the PQQ C5 atom. In this step, the product and aPQQ intermediate are Íirrrned. In the remaining steps of the reaction, the cofactor intermediate isconvertcd into reduced PQQ. For s-GDH this resolves the second controversy about thc catalyt icmechan ism.As for s-GDI-I, scveral crystal structures of PQQ-dependent methanol dehydrogenase(MDH) have heen previor.rsly deterrnined, including a complex of this cnzyme with i ts naturalsubstrate meth:inol. On the basis oÍ' this latter complex, however, a cornpletely difÍ'erent catalyticmcchanisrn has bcen proposecl, even though several l incs of biochemical and kinetic evidence arestr ikingly similar for both enzymes. To resolve this discrepancy, we have compared the twocnzynre-substrate cornplcxes in Chapter 5. In both conrplexes, PQQ, the calcium ions, the basicantino acid resiclues ancl the sl lbstrates glucose and methanol are al l  bound at similar posit ions. Thissuggcsts thi i t  MDH and s-GDH catalyze the oxidation of their substrates through an identicalreaction ntechanisnt. Al ' ter a detai led analysis of al l  structural and biochemical data we propose thatthis rnechanism proceeds as described above for s-GDH. Thus, the PQQ-dependent enzymes MDHand s-GDH usc a mechanism that is similar to that of the nicotinamide- and Í1avin-dependentenzytnes. l lence. i t  appears that hydride transÍ 'cr is a cotnmon mechanism in cnzymaticdehydrogenation reactions.1 0 8Dehydrogenases cn oxiclasesandere alcoholen oln in I l toledehydlogenases en oxidasenicotinarnides. f lavines. en cvan cle nicotinarnide- en f lavirciigeren tnet hun substraten ,naar de col 'actor. Het is echwerken.Dit proefschrift beswerkingsrlechlnisme van eernet bchulp van róntgendilïraris PQQ) in hct act ief centrumIn HooÍdstuk I wbindende eiwitten samelwerkingsmechanisme van Ponderl ing in de plaats oP de cde identiteit van het intennedDe kristallisatie, strtHoofdstuk 2. De driedimatoornbevattende kristallen.verstrooiing door het oPloskristal logral ische sYmmetriinterpreteerbare elektronendivervolgens verf i jnd kon wolvan elk 454 residuen. Beirwordt. De structuur ls nÍ(propellerbladen). die net alrdraai-as van PseudosYmmeraakvlak van de monomeren

Structure and mechanism of soluble quinoprotein glucose dehydrogenase

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Structure and mechanism of soluble quinoprotein glucose dehydrogenase

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