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Xanthine phosphoribosyltransferase (XPRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) are purine salvaging enzymes of Leishmania donovani with distinct 6-oxopurine specificities. LdXPRT phosphoribosylates xanthine, hypoxanthine, and guanine, with preference towards xanthine, whereas LdHGPRT phosphoribosylates only hypoxanthine and guanine. In our study, LdXPRT was used as a model to understand these purine base specificities. Mutating I209 to V, the conserved residue found in HGPRTs, reduced the affinity of LdXPRT for xanthine, converting it to an HGXPRT-like enzyme. The Y208F mutation in the active site indicated that aromatic residue interactions with the purine ring are limited to pi-pi binding forces and do not impart purine base specificity. Deleting the unique motif (L55-Y82) of LdXPRT affected enzyme activity. Our studies established I209 as a key residue determining the 6-oxopurine specificity of LdXPRT.

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-D-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme.

The S-adenosyl-L-methionine (SAM)-dependent methyltransferases attach a methyl group to the deprotonated methyl lysine (Kme0) using SAM as a donor. An intriguing, yet unanswered, question is how the deprotonation of the methyl lysine takes place which results in a lone pair of electrons at the Nϵ atom of the methyl lysine for the following methyl transfer. PRDM9, one of the few methyltransferases with well-defined enzyme activity in vitro and in vivo, is a good representative of the PR/SET domain methyltransferase family to study the deprotonation and subsequently the methyl transfer. The reaction consists of two progressing steps: (i) the absolutely required substrate methyl lysine deprotonation and (ii) the transfer of the methyl group to the deprontonated methyl lysine. We use empirical valence bond (EVB) simulations to evaluate Y357 at the active site as potential general base for the deprotonation of the methyl lysine. Indeed, our study has found that the pKa of Tyr357 is low enough to make it an ideal candidate for proton abstraction from the methyl lysine. The partially deprontonated Tyr357 is able to change its H-bond pattern thus bridging two proton tunneling states (OH- H 0-Tyr357 and Kme0-Nϵ H O-Tyr357) and providing a cascading proton transfer from Tyr357 to hydroxide, generating deprotonated Tyr357 and then from Kme0 to the deprotonated Tyr357 resulting in deprotonated methyl lysine. This cascading proton transfer shortens the lifespan of the labile intermediates, and affects the conformational changes during the product release important to promote the proton release to the bulk solvent. Our computational efforts have uncovered a new catalytic mechanism to unravel the unanswered question about the deprotonation of the methyl lysine in methyltransferases. (Less)

Polyhydroxyalkanoate (PHA) synthases catalyze the polymerization reaction of the acyl moiety of hydroxyacyl-coenzyme A into polyester. The catalytic subunit PhaC of PHA synthase has the PhaC box sequence at the active site that is typically described as G-X-C-X-G-G (X is an arbitrary amino acid), and cysteine is an active center. In this study, an amino acid replacement was introduced into the PhaC box of the PHA synthase derived from Ralstonia eutropha (PhaCRe ) to investigate the importance of highly conserved residues in polymerizing activity. Point mutagenesis revealed that PhaCRe mutants with the expanded PhaC box sequence ([GAST]-X-C-X-[GASV]-[GA]) are functional PHA synthases. These findings highlight the low mutational robustness of the last glycine residue in the PhaC box as well as that of the active center cysteine.

Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S-malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active-site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction. Herein, we report structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. High-resolution X-ray crystallographic results reveal three distinct FumC active-site conformations; disordered-open, ordered-open, and the newly discovered ordered-closed. Surprisingly, each SS Loop variant has unaffected Michaelis constants coupled to reductions in turnover number. Based upon our structural and functional analyses, we propose structural and catalytic roles for each of the aforementioned residues.

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (FeB ). Heme b3 and FeB constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB -less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho-acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues.

We have studied a series of human acetyl-CoA carboxylase (ACC) 1 and ACC2 proteins with deletions and/or Ser to Ala substitutions of the known phosphorylation sites. In vitro dephosphorylation/phosphorylation experiments reveal a substantial level of phosphorylation of human ACCs produced in insect cells. Our results are consistent with AMPK phosphorylation of Ser29 , Ser80 , Ser1,201 , and Ser1,216 . Phosphorylation of the N-terminal regulatory domain decreases ACC1 activity, while phosphorylation of residues in the ACC central domain has no effect. Inhibition of the activity by phosphorylation is significantly more profound at citrate concentrations below 2 mm. Furthermore, deletion of the N-terminal domain facilitates structural changes induced by citrate, including conversion of ACC dimers to linear polymers. We have also identified ACC2 amino acid mutations affecting specific inhibition of the isozyme by compound CD-017-0191. They form two clusters separated by 60-90 A: one located in the vicinity of the BC active site and the other one in the vicinity of the ACC1 phosphorylation sites in the central domain, suggesting a contribution of the interface of two ACC dimers in the polymer to the inhibitor binding site.

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (kcat ≈ 104 s-1 ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine β-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).

Chitosanases belong to glycoside hydrolase families 5, 7, 8, 46, 75 and 80 and hydrolyse glucosamine polymers produced by partial or full deacetylation of chitin. Herein, we determined the crystal structure of chitosanase from the β‐proteobacterium, Mitsuaria chitosanitabida, (McChoA) at 1.75 Å resolution; the first structure of a family 80 chitosanase. McChoA is a 34 kDa extracellular protein of 301 amino acids that fold into two (upper and lower) globular domains with an active site cleft between them. Key substrate‐binding features are conserved with family 24 lysozymes and family 46 chitosanases. The distance between catalytic residues E41 and E61 (10.8 Å) indicates an inverting type mechanism. Uniquely, three disulphide bridges and the C terminus might contribute to enzyme activity.

Mg2+ -dependent catechol-O-methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol-O-methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.

While multiple α 1-2-mannosidases are necessary for glycoprotein N-glycan maturation in vertebrates, a single bacterial α1-2-mannosidase can be sufficient to cleave all α1-2-linked mannose residues in host glycoprotein N-glycans. We report here the characterization and crystal structure of a new α1-2-mannosidase (EfMan-I) from Enterococcus faecalis, a Gram-positive opportunistic human pathogen. EfMan-I catalyzes the cleavage of α1-2-mannose from not only oligomannoses but also high-mannose-type N-glycans on glycoproteins. Its 2.15 A resolution crystal structure reveals a two-domain enzyme fold similar to other CAZy GH92 mannosidases. An unexpected potassium ion was observed bridging two domains near the active site. These findings support EfMan-I as an effective catalyst for in vitro N-glycan modification of glycoproteins with high-mannose-type N-glycans.

Serine hydroxymethyltransferase (SHMT) is the major source of 1-carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2-antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti-cancer drug target.

Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.

In this study, we investigate the inhibition of human angiogenin by ammonium sulfate. The inhibitory potency of ammonium sulfate for human angiogenin (IC50 = 123.5 ± 14.9 mm) is comparable to that previously reported for RNase A (119.0 ± 6.5 mm) and RNase 2 (95.7 ± 9.3 mm). However, analysis of two X-ray crystal structures of human angiogenin in complex with sulfate anions (in acidic and basic pH environments, respectively) indicates an entirely distinct mechanism of inhibition. While ammonium sulfate inhibits the ribonucleolytic activity of RNase A and RNase 2 by binding to the active site of these enzymes, sulfate anions bind only to peripheral substrate anion-binding subsites of human angiogenin, and not to the active site.

The Bacova_02091 gene in the β‐mannan utilization locus of Bacteroides ovatus encodes a family GH36 α‐galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterization of recombinant BoGal36A reveals unique properties compared to other GH36 α‐galactosidases, which preferentially hydrolyse terminal α‐galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (> 90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose‐substituted polymannose backbone.

EcL-DER, the aspartate/glutamate racemase from the pathogen Escherichia coli O157, exhibits racemase activity for l-aspartate and l-glutamate. This study reports the crystal structures of apo-EcL-DER, the EcL-DER-l-aspartate and the EcL-DER-d-aspartate complexes. The EcL-DER structure contains two domains, forming pseudo-mirror symmetry in the active site. A unique catalytic pair consisting of Thr(83) and Cys(197) exists in the active site. The characteristic conformations of l-Asp and d-Asp in the active site provide a straight structural evidence for the racemization mechanism of EcL-DER. In addition, the diversity of catalytic pairs implies that PLP-independent amino acid racemases adopt various catalytic mechanisms and are classified into different subgroups.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.

Peptidase-E, a nonclassical serine peptidase, is specific for dipeptides with an N-terminal aspartate. This stringent substrate specificity remains largely unexplained. We report an aspartate-bound structure of peptidase-E at 1.83 A resolution. In contrast to previous reports, the enzyme forms a dimer, and the active site is located at the dimer interface, well shielded from the solvent. Our findings further suggest that the stringent aspartate specificity of the enzyme is due to electrostatics and molecular complementarity in the active site. The new structural information presented herein may provide insights into the role of functionally important residues in peptidase-E.

Glutamate racemases (GR) catalyze the racemization of d- and l-glutamate and are targets for the development of antibiotics. We demonstrate that GR from the periodontal pathogen Fusobacterium nucleatum (FnGR) catalyzes the racemization of d-homocysteic acid (d-HCA), while l-HCA is a poor substrate. This enantioselectivity arises because l-HCA perturbs FnGR's monomer-dimer equilibrium toward inactive monomer. The inhibitory effect of l-HCA may be overcome by increasing the total FnGR concentration or by adding glutamate, but not by blocking access to the active site through site-directed mutagenesis, suggesting that l-HCA binds at an allosteric site. This phenomenon is also exhibited by GR from Bacillus subtilis, suggesting that enantiospecific, "substrate"-induced dissociation of oligomers to form inactive monomers may furnish a new inhibition strategy.

application/pdf, Article, FEBS Letter, 2018, 592, p.1378-1388

In detoxification and fermentation processes, acylating dehydrogenases catalyze the reversible oxidation of aldehydes to their corresponding acyl-CoA esters. Here, we characterize an enzyme from Aquincola tertiaricarbonis L108 responsible for prenal (3-methyl-2-butenal) to 3-methylcrotonyl-CoA oxidation. Enzyme kinetics demonstrate a preference for C5 substrates not yet observed in aldehyde dehydrogenases. Compared to acetaldehyde and acetyl-CoA, conversion of valeraldehyde and valeryl-CoA is > 100- and 8-fold more efficient, respectively. Enzyme variants with A254I, A254P, and A254G mutations indicate that active site Ala preceding the catalytic C255 is crucial for this unique specificity. These results shed new light on evolutionary adaptation of aldehyde dehydrogenases toward xenobiotics and structure-guided design of highly specific enzymes for production of biofuels, such as linear or iso-branched butanols and pentanols.

Griseoviridin (GV) is a broad-spectrum antibiotic with antibacterial and antifungal activity. In the GV biosynthetic pathway, SgvP catalyzes formation of the carbon-sulfur bond in GV. Herein, we report the recombinant expression and characterization of SgvP from Streptomyces griseoviridis NRRL2427. We also present the 2.6 Å crystal structure of SgvP, which is the first structure of a cytochrome P450 involved in carbon-sulfur bond formation in GV. Structural analysis indicates that Pro237 in the I-helix of SgvP may play a critical role in dioxygen binding and proton transfer during the catalytic cycle. Of the three channels we observed in SgvP, channel 3 may be essential for substrate ingress and egress from the active site, while channels 1 and 2 may be the solvent and water pathway, respectively.Coordinate and structure factor were deposited in the Protein Data Bank database under the accession number 4MM0.

To investigate the function of the pa4079 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1, we determined its crystal structure and confirmed it to be a NAD(P)-dependent short-chain dehydrogenase/reductase. Structural similarity and activity for a broad range of substrates indicate that PA4079 functions as a carbonyl reductase. Comparison of apo- and holo-PA4079 shows that NADP stabilizes the active site specificity loop, and small molecule binding induces rotation of the Tyr183 side chain by approximately 90° out of the active site. Quantitative real-time PCR results show that pa4079 maintains high expression levels during antibiotic exposure. This work provides a starting point for understanding substrate recognition and selectivity by PA4079, as well as its possible reduction of antimicrobial drugs.Structural data are available in the Protein Data Bank (PDB) under the following accession numbers: apo PA4079 (condition I), 5WQM; apo PA4079 (condition II), 5WQN; PA4079 + NADP (condition I), 5WQO; PA4079 + NADP (condition II), 5WQP.

We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A).

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD+ revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD+ phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors.

Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50 vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N(6)-methyladenosine (m(6)A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m(6)A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m(6)A demethylation.

Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site.

The antineoplastic target aldo–keto reductase family member 1B10 (AKR1B10) and the critical polyol pathway enzyme aldose reductase (AKR1B1) share high structural similarity. Crystal structures reported here reveal a surprising Trp112 native conformation stabilized by a specific Gln114-centered hydrogen bond network in the AKR1B10 holoenzyme, and suggest that AKR1B1 inhibitors could retain their binding affinities toward AKR1B10 by inducing Trp112 flip to result in an “AKR1B1-like” active site in AKR1B10, while selective AKR1B10 inhibitors can take advantage of the broader active site of AKR1B10 provided by the native Trp112 side-chain orientation.

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the β-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.

The crystal structure of the N-terminal putative catalytic domain of a glycoside hydrolase family 131 protein from Coprinopsis cinerea (CcGH131A) was determined. The structure of CcGH131A was found to be composed of a β-jelly roll fold and mainly consisted of two β-sheets, sheet-A and sheet-B. A concave of sheet-B, the possible active site, was wide and shallow, and three glycerol molecules were present in the concave. Arg96, Glu98, Glu138, and His218 are likely to be catalytically critical residues, and it was suggested that the catalytic mechanism of CcGH131A is different from that of typical glycosidases.

The crystal structure of metagenome-derived LC9-RNase H1 was determined. The structure-based mutational analyses indicated that the active site motif of LC9-RNase H1 is altered from DEDD to DEDN. In this motif, the location of the second glutamate residue is moved from αA-helix to β1-strand immediately next to the first aspartate residue, as in the active site of RNase H2. However, the structure and enzymatic properties of LC9-RNase H1 highly resemble those of RNase H1, instead of RNase H2. We propose that LC9-RNase H1 represents bacterial RNases H1 with an atypical DEDN active site motif, which are evolutionarily distinct from those with a typical DEDD active site motif.

N-Acetylneuraminate lyase is the first committed enzyme in the degradation of sialic acid by bacterial pathogens. In this study, we analysed the kinetic parameters of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus (MRSA). We determined that the enzyme has a relatively high KM of 3.2 mM, suggesting that flux through the catabolic pathway is likely to be controlled by this enzyme. Our data indicate that sialic acid alditol, a known inhibitor of N-acetylneuraminate lyase enzymes, is a stronger inhibitor of MRSA N-acetylneuraminate lyase than of Clostridium perfringens N-acetylneuraminate lyase. Our analysis of the crystal structure of ligand-free and inhibitor-bound MRSA N-acetylneuraminate lyase suggests that subtle dynamic differences in solution and/or altered binding interactions within the active site may account for species-specific inhibition. This article is protected by copyright. All rights reserved.

The characteristic His-Glu catalytic dyad of the disulfide oxidoreductase (DSOR) family of enzymes is replaced in 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase (2-KPCC) by the residues Phe-His. 2-KPCC is the only known carboxylating member of the DSOR family and has replaced this dyad potentially to eliminate proton-donating groups at a key position in the active site. Substitution of the Phe-His by the canonical residues results in production of higher relative concentrations of acetone versus the natural product acetoacetate. The results indicate that these differences in 2-KPCC are key to discriminating between carbon dioxide and protons as attacking electrophiles. This article is protected by copyright. All rights reserved.

Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, OxyA, escaping structural analysis. We are now able to present the structure of OxyAtei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active OxyA homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of OxyAtei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.

When bound to ADP, ATP-dependent protease FtsH subunits adopt either an “open” or “closed” conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lid-like helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH. Structured summary of protein interactions FtsH and FtsH bind by molecular sieving ( View Interaction )

rv1098c, an essential gene in Mycobacterium tuberculosis, codes for a class II fumarase. We describe here the crystal structure of Rv1098c in complex with l -malate, fumarate or the competitive inhibitor meso-tartrate. The models reveal that substrate binding promotes the closure of the active site through conformational changes involving the catalytic SS-loop and the C-terminal domain, which likely represents a general feature of this enzyme superfamily. Analysis of ligand–enzyme interactions as well as site-directed mutagenesis suggest Ser318 as one of the two acid–base catalysts. Structured summary of protein interactions Rv1098c and Rv1098c bind by X-ray crystallography ( View interaction )

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic α-amylase we describe the chemoenzymatic synthesis of the disaccharide analog α-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic α-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.

Covalent, mechanism-based inhibitors of glycosidases are valuable probe molecules for visualising enzyme activities in complex systems. We here describe the chemoenzymatic synthesis of 6-phospho-cyclophellitol and evaluate its behaviour as a mechanism-based inactivator of the Streptococcus pyogenes 6-phospho-β-glucosidase from CAZy family GH1. We further present the three-dimensional structure of the inactivated enzyme, which reveals the constellation of active site residues responsible for the enzyme's specificity and confirms the covalent nature of the inactivation. This article is protected by copyright. All rights reserved.

To investigate ligand selectivity between the oncogenic KDM4C and tumor repressor protein KDM6A histone demethylases, KDM4C and KDM6A were enzymatically characterized, and subsequently, four compounds were tested for inhibitory effects. 2,4-dicarboxypyridine and (R)-N-oxalyl-O-benzyltyrosine (3) are both known to bind to a close KDM4C homolog and 3 binds in the part of the cavity that accommodates the side chain in position 11 of histone 3. The inhibition measurements showed significant selectivity between KDM4C and KDM6A. This demonstrates that despite very similar active site topologies, selectivity between Jumonji family histone demethylases can be obtained even with small molecule ligands.

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS-b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS-b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α-farnesene, can also synthesize the monoterpene (E)-β-ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS-b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non-synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS-b enzymes.

International audience; Trx-z is a chloroplastic thioredoxin, exhibiting a usual WCGPC active site, but whose biochemical properties are unknown. We demonstrate here that Trx-z supports the activity of several plastidial antioxidant enzymes, such as thiol-peroxidases and methionine sulfoxide reductases, using electrons provided by ferredoxin-thioredoxin reductase. Its disulfide reductase activity requires the presence of both active site cysteines forming a catalytic disulfide bridge with a midpoint redox potential of -251 mV at pH7. These in vitro biochemical data suggest that, besides its decisive role in the regulation of plastidial transcription, Trx-z might also be involved in stress response.