Isolation and characterization of a bis(dithiolene)-supported tungsten-acetylenic complex as a model for acetylene hydratase.

Acetylene hydratase is currently the only known mononuclear tungstoenzyme that does not catalyze a net redox reaction. The conversion of acetylene to acetaldehyde is proposed to occur at a W(IV) active site through first-sphere coordination of the acetylene substrate. To date, a handful of tungsten complexes have been shown to bind acetylene, but many lack the bis(dithiolene) motif of the native enzyme. The model compound, [W(O)(mnt)2]2-, where mnt2- is 1,2-dicyano-1,2-dithiolate, was previously reported to bind an electrophilic acetylene substrate, dimethyl acetylenedicarboxylate, and characterized by FT-IR, UV-vis, potentiometry, and mass spectrometry (Yadav, J; Das, S. K.; Sarkar, S., J. Am. Chem. Soc., 1997, 119, 4316-4317). By slightly changing the electrophilic acetylene substrate, an acetylenic-bis(dithiolene)­tungsten(IV) complex has been isolated and characterized by FT-IR, UV-vis, NMR, X-ray diffraction, and X-ray absorption spectroscopy. Activation parameters for complex formation were also determined and suggest coordination-sphere reorganization is a limiting factor in the model complex reactivity.

HadBD dehydratase from Mycobacterium tuberculosis fatty acid synthase type II: A singular structure for a unique function.

Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBDMtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBDMtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBDMtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex.

Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

Structural and Biochemical Analysis of 3-Dehydroquinate Dehydratase from Corynebacterium glutamicum.

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQDWT with the citrate at a resolution of 1.80Å and CgDHQDR19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQDS103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQDS103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

Exploring the Substrate-Assisted Dehydration of Chorismate Catalyzed by Dehydratase MqnA from QM/MM Calculations: The Role of Pocket Residues and the Hydrolysis Mechanism of N17D Mutant.

MqnA is the first enzyme on the futalosine pathway to menaquinone, which catalyzes the dehydration of chorismate to yield 3-enolpyruvyl-benzoate (3-EPB). MqnA is also the only chorismate dehydratase known so far. In this work, based on the recently determined crystal structures, we constructed the enzyme-substrate complex models and conducted quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the reaction details of MqnA and the critical roles of pocket residues. The calculation results confirm that the MqnA-catalyzed dehydration of chorismate follows the substrate-assisted E1cb mechanism, in which the enol carboxylate in the side chain of the substrate is responsible for deprotonating the C3 of chorismate. This proton transfer process is much slower than C4-OH departure. Calculations on different mutants reveal that S86 and N17 are important for anchoring the enol carboxylate of the substrate in a favorable conformation to extract the C3-proton. The strong H-bonds formed between the enol carboxylate of chorismate and S86/N17 play a key role in stabilizing the reaction intermediate. Consistent with the experimental observations, our calculations demonstrate that the MqnA N17D mutant also shows hydrolase activity and the typical enzyme-catalyzed hydrolysis mechanism is elucidated. The protonated D17 is responsible for saturating the methylene group of chorismate to start the hydrolysis reaction. The orientation of the carboxyl group of D17 is key in determining MqnA to be a dehydratase or hydrolase.

A nanobiocatalytic system based on the self-assembly of ferrous phosphate with a high regioselectivity nitrile hydratase.

Our previous study identified a novel nitrile hydratase (NHase) with remarkable biotransformation activity toward adipamide during the production of 5-cyanovaleramide (5-CVAM), an important intermediate of herbicide and chemical raw material. Nevertheless, free NHase will face harsh conditions if they are applied directly in industrial processes. In this study, we, therefore, prepared Fe3(PO4)2 hybrid nanoflowers for NHase immobilization based on the protein-inorganic hybrid self-assembly by establishing a novel and facile method. The results showed that the NHase@Fe3(PO4)2 nanoflowers had significantly enhanced tolerance to the temperature ranging from 40°C to 60°C when compared with free NHase. The catalytic activity of NHase@Fe3(PO4)2 nanoflowers remained high in extreme pH environments such as weak acid (pH 5) and strong alkali (pH 10) environments. In addition, the storage stability and reusability of encapsulated NHase were also superior to that of free NHase. NHase@Fe3(PO4)2 nanoflowers had a notable feature of high substrate tolerance. We found NHase@Fe3(PO4)2 nanoflowers still had 65% activity as the adiponitrile concentration increased up to 200 mmol L-1, whereas free NHase almost lost their catalytic activity when the adiponitrile concentration was just 100 mmol L-1. All of these results clearly demonstrated that ferrous phosphate nanocrystals might offer a novel strategy for 5-CVAM production with nanobiocatalytic systems.

Structural and Functional Insight into the Mechanism of the Fe-S Cluster-Dependent Dehydratase from Paralcaligenes ureilyticus.

Enzyme-catalyzed reaction cascades play an increasingly important role for the sustainable manufacture of diverse chemicals from renewable feedstocks. For instance, dehydratases from the ilvD/EDD superfamily have been embedded into a cascade to convert glucose via pyruvate to isobutanol, a platform chemical for the production of aviation fuels and other valuable materials. These dehydratases depend on the presence of both a Fe-S cluster and a divalent metal ion for their function. However, they also represent the rate-limiting step in the cascade. Here, catalytic parameters and the crystal structure of the dehydratase from Paralcaligenes ureilyticus (PuDHT, both in presence of Mg2+ and Mn2+ ) were investigated. Rate measurements demonstrate that the presence of stoichiometric concentrations Mn2+ promotes higher activity than Mg2+ , but at high concentrations the former inhibits the activity of PuDHT. Molecular dynamics simulations identify the position of a second binding site for the divalent metal ion. Only binding of Mn2+ (not Mg2+ ) to this site affects the ligand environment of the catalytically essential divalent metal binding site, thus providing insight into an inhibitory mechanism of Mn2+ at higher concentrations. Furthermore, in silico docking identified residues that play a role in determining substrate binding and selectivity. The combined data inform engineering approaches to design an optimal dehydratase for the cascade.

Molecular Insights into Bifunctional Ambruticin DH3 for Substrate Specificity and Catalytic Mechanism.

Dehydratase (DH), a domain located at polyketide synthase (PKS) modules, commonly catalyzes the dehydration of ß-hydroxy to an α,ß-unsaturated acyl intermediate. As a unique bifunctional dehydratase, AmbDH3 (the DH domain of module 3 of the ambruticin PKS) is verified to be responsible for both dehydration and the following pyran-forming cyclization. Besides, in vitro studies showed that its catalytic efficiency varies with different chiral substrates. However, the detailed molecular mechanism of AmbDH3 remains unclear. In this work, the structural rationale for the substrate specificity (2R/2S- and 6R/6S-substrates) in AmbDH3 was elucidated and the complete reaction pathways including dehydration and cyclization were presented. Both MD simulations and binding free energy calculations indicated AmbDH3 had a stronger preference for 2R-substrates (2R6R-2, 2R6S-3) than 2S-substrates (2S6R-1), and residue H51 and G61 around the catalytic pocket were emphasized by forming stable hydrogen bonds with 2R-substrates. In addition, AmbDH3's mild tolerance at C6 was explained by comparison of substrate conformation and hydrogen bond network in 6S- and 6R-substrate systems. The QM/MM results supported a consecutive one-base dehydration and cyclization mechanism for 2R6S-3 substrate with the energy barrier of 25.2 kcal mol-1 and 24.5 kcal mol-1 , respectively. Our computational results uncover the substrate recognition and catalytic process of the first bifunctional dehydratase-cyclase AmbDH3, which will shed light on the application of multifunctional DH domains in PKSs for diverse natural product analogs and benefit the chemoenzymatic synthesis of stereoselective pyran-containing products.

1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.

Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus (S.t NHase) was successfully overexpressed in Escherichia coli. The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant ßL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into ßLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a "toolbox" containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.

Bioinspired Iron-Catalyzed Dehydration of Aldoximes to Nitriles: A General N-O Redox-Cleavage Method.

Inspired by OxdA that operates biocatalytic aldoxime dehydration, we have developed an efficient iron catalyst, Cp*Fe(1,2-Cy2PC6H4O) (1), which rapidly converts various aliphatic and aromatic aldoximes to nitriles with release of H2O at room temperature. The catalysis involves redox activation of the N-O bond by a 1e- transfer from the iron catalyst to the oxime. Such redox-mediated N-O cleavage was demonstrated by the isolation of a ferrous iminato intermediate from the reaction of the ketoxime substrate. This iron-catalyzed acceptorless dehydration approach represents a general method for the preparation of nitriles, and it also delivers salicylonitriles by catalyzing the Kemp elimination reaction.

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and L-aspartate-ß-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml-1 DHDPS. The crystals diffracted to 2.15 Šresolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the ß-δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

Discovery of the ATPase Activity of a Cobalt-Type Nitrile Hydratase Activator and Its Promoting Effect on Enzyme Maturation.

An activator protein and a metal ion are two factors known to be indispensable for the maturation of nitrile hydratase (NHase). Here, the third key factor, adenosine triphosphate (ATP), was identified to play an important role in the activation of Co-type NHase. Free phosphate measurements revealed that the Co-type activator protein can hydrolyze ATP/GTP with appreciable performance and that such catalytic performance is related to NHase activity. Computational analysis and site-directed mutagenesis identified several potential hot spot residues involved in the binding of ATP to Co-type activator protein, and an E60A/W61A/D62A/I139A/T141A combinatorial variant reduced the ATPase activity to 18% of its original level. Further NHase activation studies using the combinatorial variant demonstrated that although the ATPase activity of the Co-type activator protein correlated with NHase activity, a low ATP concentration of 0.5 mmol/L was optimal for NHase activation, with higher ATP concentrations potentially inhibiting NHase activity.

Functional and ligand binding studies of NAD(P)H hydrate dehydratase enzyme from vancomycin-resistant Staphylococcus aureus by NMR spectroscopic approach, including saturation transfer difference (STD-NMR) spectroscopy.

NADH and NADPH are labile coenzymes that undergo hydration by enzymatic reaction or by heat at 5,6 double bond, and convert into non-functional hydrates, NADHX and NADPHX, respectively. The NAD(P)H hydrate dehydratase enzyme catalyzes the dehydration of S-NADHX/S-NADPHX at the expense of ATP, and thus contributes in the nicotinamide nucleotide repair process. This enzyme is also known as "metabolite-proofreading enzyme". Herein, we report the molecular cloning and expression of this highly conserved enzyme of vancomycin-resistant Staphylococcus aureus (VRSA). Its functional and inhibition studies were performed for the first time by NMR spectroscopy. NMR studies showed the dehydration of S epimer of NADHX, in the presence of R-NADHX and cyc-NADHX, by NAD(P)H hydrate dehydratase. In addition, by employing the STD-NMR approach, a library of drugs and natural products (total 79) were evaluated for their binding interactions with the NAD(P)H hydrate dehydratase enzyme. Among them, seven compounds showed ligand-like interactions with the enzyme, and thus functional activity of the enzyme was again checked in the presence of each ligand. Compound 2 (Thiamine HCl) was found to fully inhibit the enzyme's function, and recognized as a potential inhibitor. Current study demonstrates that this enzyme deserves further studies as a potential drug target, as its inhibition can disrupt the normal metabolism of pathogenic VRSA.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Towards an understanding of oleate hydratases and their application in industrial processes.

Fatty acid hydratases are unique to microorganisms. Their native function is the oxidation of unsaturated C-C bonds to enable detoxification of environmental toxins. Within this enzyme family, the oleate hydratases (Ohys), which catalyze the hydroxylation of oleic acid to 10-(R)-hydroxy stearic acid (10-HSA) have recently gained particular industrial interest. 10-HSA is considered to be a replacement for 12-(R)-hydroxy stearic acid (12-HSA), which has a broad application in the chemical and pharmaceutical industry. As 12-HSA is obtained through an energy consuming synthesis process, the biotechnological route for sustainable 10-HSA production is of significant industrial interest. All Ohys identified to date have a non-redox active FAD bound in their active site. Ohys can be divided in several subfamilies, that differ in their oligomerization state and the decoration with amino acids in their active sites. The latter observation indicates a different reaction mechanism across those subfamilies. Despite intensive biotechnological, biochemical and structural investigations, surprising little is known about substrate binding and the reaction mechanism of this enzyme family. This review, summarizes our current understanding of Ohys with a focus on sustainable biotransformation.

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli.

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.

Nucleotide sugar dehydratases: Structure, mechanism, substrate specificity, and application potential.

Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies.

Altering the binding determinant on the interdigitating loop of mandelate racemase shifts specificity towards that of d-tartrate dehydratase.

The enolase superfamily (ENS) has served as a paradigm for understanding how enzymes that share a conserved structure, as well as a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation), evolved from a common progenitor to catalyze mechanistically diverse reactions. Enzymes of the mandelate racemase (MR)-subgroup of the ENS share interdigitating loops between adjacent, 2-fold symmetry-related protomers of the tightly associated homodimers that comprise their quaternary structures. For the MR-subgroup members MR and d-tartrate dehydratase (TarD), the tip of the loop contributes a binding determinant to the adjacent active site (i.e., Leu 93 and Lys 102, respectively). To assess the role of Leu 93 of MR in substrate specificity and catalysis, we constructed L93 variants bearing hydrophobic (L93A, L93F, and L93W), polar neutral (L93N), acidic (L93D), or basic (L93K and L93R) residues at position 93. Gel filtration-HPLC revealed that wild-type MR and all L93 MR variants, apart from L93R MR (dimeric), were tetrameric in solution. The catalytic efficiency (kcat/Km) was reduced in the R→S and S→R reaction directions for all variants, primarily due to reduced turnover (kcat). Substitution of Leu 93 by Lys or Arg to mimic Lys 102 of TarD enhanced the binding of malate and tartrate, with meso- and d-tartrate exhibiting linear mixed-type inhibition of L93K MR. Despite the striking 500-fold increase in the binding affinity of d-tartrate, relative to (S)-mandelate, L93K MR exhibited no TarD activity. MD simulations suggested that the failure of L93K MR to catalyze α-deprotonation (i.e., H-D exchange) arises from inappropriate positioning of the Brønsted base (Lys 166). Thus, a change in binding determinant on the interdigitating loop can play a significant role in governing substrate specificity within the ENS, but does not necessarily confer 'new' catalytic activity despite similarities in catalytic machinery.

The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5'-deoxyadenosine and not 5'-deoxy-5'-(methylthio)adenosine.

Glycerol dehydratase activating enzyme (GD-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential amino acid backbone radical onto glycerol dehydratase in bacteria under anaerobic conditions. Although GD-AE is closely homologous to other radical SAM activases that have been shown to cleave the S-C(5') bond of SAM to produce 5'-deoxyadenosine (5'-dAdoH) and methionine, GD-AE from Clostridium butyricum has been reported to instead cleave the S-C(γ) bond of SAM to yield 5'-deoxy-5'-(methylthio)adenosine (MTA). Here we re-investigate the SAM cleavage reaction catalyzed by GD-AE and show that it produces the widely observed 5'-dAdoH, and not the less conventional product MTA.