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

Insights into the Substrate Specificity of Archaeal Entner-Doudoroff Aldolases:The Structures of Picrophilus torridus 2-Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2-Keto-3-deoxy-6-phosphogluconate Aldolase in Complex with 2-Keto-3-deoxy-6-phosphogluconate

The thermoacidophilic archaea <i>Picrophilus torridus</i> and <i>Sulfolobus solfataricus</i> catabolize glucose via a nonphosphorylative Entner–Doudoroff pathway and a branched Entner–Doudoroff pathway, respectively. Key enzymes for these Entner–Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD­(P)­G-aldolase]. KDG-aldolase from <i>P. torridus</i> (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD­(P)­G-aldolase from <i>S. solfataricus</i> [Ss-KD­(P)­G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD­(P)­G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD­(P)­G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from <i>P. torridus</i> at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD­(P)­G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage

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/

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

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

The synthesis and kinetic evaluation of aryl α-aminophosphonates as novel inhibitors of T. cruzi trans-sialidase

International audienceThe trans-sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle of this human pathogenic parasite and is considered a promising target for the development of new drug treatments against Chagas' disease. Here we describe α-amino phosphonates as a novel class of inhibitor of T. cruzi trans-sialidase. Molecular modelling studies were initially used to predict the active-site binding affinities for a series of amino phosphonates, which were subsequently synthesised and their IC50s determined in vitro. The measured inhibitory activities show some correlation with the predictions from molecular modelling, with 1-napthyl derivatives found to be the most potent inhibitors having IC50s in the low micromolar range. Interestingly, kinetic analysis of the mode of inhibition demonstrated that the α-aminophosphonates tested here operate in a non-competitive manner