Structurally-informed Mutagenesis of a Stereochemically Promiscuous Aldolase Produces Mutants that Catalyse the Diastereoselective Syntheses of all Four Stereoisomers of 3-Deoxy-Hexulosonic acid

[Image: see text] A 2-keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus catalyzes the nonstereoselective aldol reaction of pyruvate and d-glyceraldehyde to produce 2-keto-3-deoxygluconate (d-KDGlc) and 2-keto-3-deoxy-d-galactonate (d-KDGal). Previous investigations into curing the stereochemical promiscuity of this hyperstable aldolase used high-resolution structures of the aldolase bound to d-KDGlc or d-KDGal to identify critical amino acids involved in substrate binding for mutation. This structure-guided approach enabled mutant variants to be created that could stereoselectively catalyze the aldol reaction of pyruvate and natural d-glyceraldehyde to selectively afford d-KDGlc or d-KDGal. Here we describe the creation of two further mutants of this Sulfolobus aldolase that can be used to catalyze aldol reactions between pyruvate and non-natural l-glyceraldehyde to enable the diastereoselective synthesis of l-KDGlc and l-KDGal. High-resolution crystal structures of all four variant aldolases have been determined (both unliganded and liganded), including Variant 1 with d-KDGlc, Variant 2 with pyruvate, Variant 3 with l-KDGlc, and Variant 4 with l-KDGal. These structures have enabled us to rationalize the observed changes in diastereoselectivities in these variant-catalyzed aldol reactions at a molecular level. Interestingly, the active site of Variant 4 was found to be sufficiently flexible to enable catalytically important amino acids to be replaced while still retaining sufficient enzymic activity to enable production of l-KDGal

Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in <em>Geobacillus thermoglucosidasius </em>

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcoholviaan aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD+pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophileGeobacillus thermoglucosidasiushas been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain.In silicomodelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.</jats:p

A novel β-xylosidase structure from Geobacillus thermoglucosidasius:The first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.</jats:p

Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophos­phate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, which can then be synthesized and experimentally characterized. Through ASR a novel PDC was generated, designated ANC27, that shares only 78% amino-acid sequence identity with its closest extant homologue (Komagataeibacter medellinensis PDC, GenBank accession No. WP_014105323.1), yet is fully functional. Crystals of this PDC diffracted to 3.5 Å resolution. The data were merged in space group P3221, with unit-cell parameters a = b = 108.33, c = 322.65 Å, and contained two dimers (two tetramer halves) in the asymmetric unit. The structure was solved by molecular replacement using PDB entry 2wvg as a model, and the final R values were Rwork = 0.246 (0.3671 in the highest resolution bin) and Rfree = 0.319 (0.4482 in the highest resolution bin). Comparison with extant bacterial PDCs supports the previously observed correlation between decreased tetramer interface area (and number of interactions) and decreased thermostability

Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by lipoate protein ligase (LplA) or by lipoic acid synthetase (LipA) and lipoyl(octanoyl) transferase (LipB) combined. Whereas bacterial and eukaryotic LplAs comprise a single, two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The structure of Thermoplasma acidophilum LplA-N is known, but the structure of LplA-C and its role in lipoylation are unknown. We have determined the structures of the substrate-free LplA-N+LplA-C complex and the dihydrolipoyl acyltransferase lipoyl domain (E2lipD) that is lipoylated by LplA-N+LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: LplA-C is disordered but folds upon association with LplA-N; LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; the adenylate binding region of LplA-N+LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; LplA-N+LplA-C and E2lipD do not interact in the absence of substrate; LplA-N+LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; LplA-N+LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.<br/

Why are the 2-oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex

The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.</jats:p

Insights into the structure-function relationships of dimeric C3d fragments

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future

Structural studies on nonlinear optical materials

This thesis reports the synthesis and structural characteristics of solid solutions derived from KTP,KTiOPO4 (space group Pn2la, Z=8, a=12.814(6)Å, b=10.616(5)Å, c= 6.404(2)Å) with substitution of K by Na and Rb; Ti by Sn, Ge and Cr; and P by As. The KTiOPO4 structure contains two formula units per asymmetric unit and hence two crystallographically distinct K, Ti and P sites. This makes substituent ordering possible with selective modification of the nonlinear optical properties. K0.5Na0.5TiOPO4, (occupancy K(1):96(7)%Na, K(2):105(8)%K) was characterised by Rietveld analysis of combined time-of-flight (TOP) neutron and X-ray powder diffraction, the X-ray diffraction data being required to distinguish between K and Na. Constant wavelength neutron powder diffraction data were used to refine the Na0.5Rb0.5TiOPO4 structure (K(1):97(3)%Na, K(2):96(3)%Rb). The cations in both these materials were fully ordered over the two possible K sites, while in K0.5Rb0.5TiOPO4 (K(1):71(2)%K, K(2):71(2)%Rb) the cation distribution, determined from a TOP neutron powder diffraction study, was only partially ordered. A single crystal X-ray diffraction study of an ion-exchanged K1-xNaxTiOPO4 sample (x=0.58), confirmed that the cation distribution observed in the powder diffraction analysis was also present after ion-exchange at lower temperatures (350°C), (K(1):93.3(1)%Na, K(2):77.3(7)%K). An ion-exchanged crystal that had been annealed at a temperature closer to the KTiOPO4 synthesis temperature gave almost identical results (K(1):94.4(6)%Na,K(2):81.0(4)%K). Substitution for Ti by Sn or Ge gave rise to only partial ordering. The structures of KTi0.5Sn0.5OPO4(Ti(1):63.9%Ti, Ti(2):64.6(5)%Sn) and KTi0.5Ge0.5OPO4 (Ti(1):76.7(4)%Ti, Ti(2):66.1(5)%Ge) were determined through combined analysis of X-ray and TOP neutron powder diffraction data. The X-ray data were required to define the Ti(1) site as the average weighted neutron scattering length was approximately zero. An apparently random distribution of As over the two P sites was demonstrated by time-of-flight powder neutron diffraction from KTiOP0.5As0.5O4(P(1):56(1)%P, P(2):56(1)%As). Measurements on KTiOPAsxO4 powders indicated that the Nd:YAG second harmonic intensity increased with As. The structures of RbTi0.5Sn0.5OPO4 and RbTiOP0.5As0.5O4 were investigated by powder X-ray diffraction and showed similar ordering to the K isomorphs. Simultaneous substitution for both K and Ti was investigated by Rietveld analysis of combined X-ray and TOP neutron powder diffraction data on K0.5Na0.5Ti0.5Sn0.5OPO4 (K(1):73(4.)%Na,Ti(1):66.9(3)%Ti), Na0.5Rb0.5Ti0.5Sn0.5OPO4 (K(1):54(2)%Rb, Ti(1):67.0(5)%Ti), K0.5Rb0.5Ti0.5Sn0.5OPO4 (K(1):77(1)%K, Ti(1):66.1(3)%Ti) and K0.5Rb0.5SnOPO4(K(1):64(2)%K). Cr-doped materials were studied by single crystal X-ray and TOP neutron powder diffraction. Doping a small quantity of Cr into KTP produced the KTi.95Cr.05OPO4 isomorph, (3.2(6)%Cr on both Ti sites). Substitution of a larger amount of Cr produced K1.8Ti1.2Cr.8(PO4)3, (space group P2l3, a=9.7902(1)Å, Ti(1):69.4(6)%Ti, Ti(2):54.9(6)%Ti) isomorphous with Langbeinite. Magnetic measurements using a SQUID susceptometer found a value for the moment consistent with Cr(III)

Structural studies on nonlinear optical materials

This thesis reports the synthesis and structural characteristics of solid solutions derived from KTP,KTiOPO4 (space group Pn2la, Z=8, a=12.814(6)Å, b=10.616(5)Å, c= 6.404(2)Å) with substitution of K by Na and Rb; Ti by Sn, Ge and Cr; and P by As. The KTiOPO4 structure contains two formula units per asymmetric unit and hence two crystallographically distinct K, Ti and P sites. This makes substituent ordering possible with selective modification of the nonlinear optical properties. K0.5Na0.5TiOPO4, (occupancy K(1):96(7)%Na, K(2):105(8)%K) was characterised by Rietveld analysis of combined time-of-flight (TOP) neutron and X-ray powder diffraction, the X-ray diffraction data being required to distinguish between K and Na. Constant wavelength neutron powder diffraction data were used to refine the Na0.5Rb0.5TiOPO4 structure (K(1):97(3)%Na, K(2):96(3)%Rb). The cations in both these materials were fully ordered over the two possible K sites, while in K0.5Rb0.5TiOPO4 (K(1):71(2)%K, K(2):71(2)%Rb) the cation distribution, determined from a TOP neutron powder diffraction study, was only partially ordered. A single crystal X-ray diffraction study of an ion-exchanged K1-xNaxTiOPO4 sample (x=0.58), confirmed that the cation distribution observed in the powder diffraction analysis was also present after ion-exchange at lower temperatures (350°C), (K(1):93.3(1)%Na, K(2):77.3(7)%K). An ion-exchanged crystal that had been annealed at a temperature closer to the KTiOPO4 synthesis temperature gave almost identical results (K(1):94.4(6)%Na,K(2):81.0(4)%K). Substitution for Ti by Sn or Ge gave rise to only partial ordering. The structures of KTi0.5Sn0.5OPO4(Ti(1):63.9%Ti, Ti(2):64.6(5)%Sn) and KTi0.5Ge0.5OPO4 (Ti(1):76.7(4)%Ti, Ti(2):66.1(5)%Ge) were determined through combined analysis of X-ray and TOP neutron powder diffraction data. The X-ray data were required to define the Ti(1) site as the average weighted neutron scattering length was approximately zero. An apparently random distribution of As over the two P sites was demonstrated by time-of-flight powder neutron diffraction from KTiOP0.5As0.5O4(P(1):56(1)%P, P(2):56(1)%As). Measurements on KTiOPAsxO4 powders indicated that the Nd:YAG second harmonic intensity increased with As. The structures of RbTi0.5Sn0.5OPO4 and RbTiOP0.5As0.5O4 were investigated by powder X-ray diffraction and showed similar ordering to the K isomorphs. Simultaneous substitution for both K and Ti was investigated by Rietveld analysis of combined X-ray and TOP neutron powder diffraction data on K0.5Na0.5Ti0.5Sn0.5OPO4 (K(1):73(4.)%Na,Ti(1):66.9(3)%Ti), Na0.5Rb0.5Ti0.5Sn0.5OPO4 (K(1):54(2)%Rb, Ti(1):67.0(5)%Ti), K0.5Rb0.5Ti0.5Sn0.5OPO4 (K(1):77(1)%K, Ti(1):66.1(3)%Ti) and K0.5Rb0.5SnOPO4(K(1):64(2)%K). Cr-doped materials were studied by single crystal X-ray and TOP neutron powder diffraction. Doping a small quantity of Cr into KTP produced the KTi.95Cr.05OPO4 isomorph, (3.2(6)%Cr on both Ti sites). Substitution of a larger amount of Cr produced K1.8Ti1.2Cr.8(PO4)3, (space group P2l3, a=9.7902(1)Å, Ti(1):69.4(6)%Ti, Ti(2):54.9(6)%Ti) isomorphous with Langbeinite. Magnetic measurements using a SQUID susceptometer found a value for the moment consistent with Cr(III).</p