Mandelate Racemase Research Articles

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.

A Phenylboronic Acid-Based Transition State Analogue Yields Nanomolar Inhibition of Mandelate Racemase.

Mandelate racemase (MR) catalyzes the Mg2+-dependent interconversion of (R)- and (S)-mandelate by stabilizing the altered substrate in the transition state (TS) by ∼26 kcal/mol. The enzyme has been employed as a model to explore the limits to which the free energy of TS stabilization may be captured by TS analogues to effect strong binding. Herein, we determined the thermodynamic parameters accompanying binding of a series of bromo-, chloro-, and fluoro-substituted phenylboronic acids (PBAs) by MR and found that binding was predominately driven by favorable entropy changes. 3,4-Dichloro-PBA was discovered to be the most potent inhibitor yet identified for MR, binding with a Kdapp value of 11 ± 2 nM and exceeding the binding of the substrate by ∼72,000-fold. The ΔCp value accompanying binding (-488 ± 18 cal·mol-1 K-1) suggested that dispersion forces contribute significantly to the binding. The pH-dependence of the inhibition revealed that MR preferentially binds the anionic, tetrahedral form of 3,4-dichloro-PBA with a pH-independent Ki value of 5.7 ± 0.5 nM, which was consistent with the observed upfield shift of the 11B NMR signal. The linear free energy relationship between log(kcat/Km) and log(1/Ki) for wild-type and 11 MR variants binding 3,4-dichloro-PBA had a slope of 0.8 ± 0.2, indicating that MR recognizes the inhibitor as an analogue of the TS. Hence, halogen substitution may be utilized to capture additional free energy of TS stabilization arising from dispersion forces to enhance the binding of boronic acid inhibitors by MR.

Capturing the free energy of transition state stabilization: insights from the inhibition of mandelate racemase.

Mandelate racemase (MR) catalyses the Mg2+-dependent interconversion of (R)- and (S)-mandelate. To effect catalysis, MR stabilizes the altered substrate in the transition state (TS) by approximately 26 kcal mol-1 (-ΔGtx), such that the upper limit of the virtual dissociation constant of the enzyme-TS complex is 2 × 10-19 M. Designing TS analogue inhibitors that capture a significant amount of ΔGtx for binding presents a challenge since there are a limited number of protein binding determinants that interact with the substrate and the structural simplicity of mandelate constrains the number of possible isostructural variations. Indeed, current intermediate/TS analogue inhibitors of MR capture less than or equal to 30% of ΔGtx because they fail to fully capitalize on electrostatic interactions with the metal ion, and the strength and number of all available electrostatic and H-bond interactions with binding determinants present at the TS. Surprisingly, phenylboronic acid (PBA), 2-formyl-PBA, and para-chloro-PBA capture 31-38% of ΔGtx. The boronic acid group interacts with the Mg2+ ion and multiple binding determinants that effect TS stabilization. Inhibitors capable of forming multiple interactions can exploit the cooperative interactions that contribute to optimum binding of the TS. Hence, maximizing interactions with multiple binding determinants is integral to effective TS analogue inhibitor design. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Application of circular dichroism-based assays to racemases and epimerases: Recognition and catalysis of reactions of chiral substrates by mandelate racemase.

Racemases and epimerases have attracted much interest because of their astonishing ability to catalyze the rapid α-deprotonation of carbon acid substrates with high pKa values (∼13-30) leading to the formation of d-amino acids or various carbohydrate diastereomers that serve important roles in both normal physiology and pathology. Enzymatic assays to measure the initial rates of reactions catalyzed by these enzymes are discussed using mandelate racemase (MR) as an example. For MR, a convenient, rapid, and versatile circular dichroism (CD)-based assay has been used to determine the kinetic parameters accompanying the MR-catalyzed racemization of mandelate and alternative substrates. This direct, continuous assay permits real time monitoring of reaction progress, the rapid determination of initial velocities, and immediate recognition of anomalous behaviors. MR recognizes chiral substrates primarily through interactions of the phenyl ring of (R)- or (S)-mandelate with the hydrophobic R- or S-pocket at the active site, respectively. During catalysis, the carboxylate and α-hydroxyl groups of the substrate remain fixed in place through interactions with the Mg2+ ion and multiple H-bonding interactions, while the phenyl ring moves between the R- and S-pockets. The minimal requirements for the substrate appear to be the presence of a glycolate or glycolamide moiety, and a hydrophobic group of limited size that can stabilize the carbanionic intermediate through resonance or strong inductive effects. Similar CD-based assays may be applied to determine the activity of other racemases or epimerases with proper consideration of the molar ellipticity, wavelength, overall absorbance of the sample, and the light pathlength.

Design and evaluation of substrate-product analoginhibitors for racemases and epimerases utilizing a 1,1-proton transfer mechanism.

Racemases and epimerases catalyze the inversion of stereochemistry at asymmetric carbon atoms to generate stereoisomers that often play important roles in normal and pathological physiology. Consequently, there is interest in developing inhibitors of these enzymes for drug discovery. A strategy for the rational design of substrate-product analog (SPA) inhibitors of racemases and epimerases utilizing a direct 1,1-proton transfer mechanism is elaborated. This strategy assumes that two groups on the asymmetric carbon atom remain fixed at active-site binding determinants, while the hydrogen and third, motile group move during catalysis, with the latter potentially traveling between an R- and S-pocket at the active site. SPAs incorporate structural features of the substrate and product, often with geminal disubstitution on the asymmetric carbon atom to simultaneously present the motile group to both the R- and S-pockets. For racemases operating on substrates bearing three polar groups (glutamate, aspartate, and serine racemases) or with compact, hydrophobic binding pockets (proline racemase), substituent motion is limited and the design strategy furnishes inhibitors with poor or modest binding affinities. The approach is most successful when substrates have a large, motile hydrophobic group that binds at a plastic and/or capacious hydrophobic site. Potent inhibitors were developed for mandelate racemase, isoleucine epimerase, and α-methylacyl-CoA racemase using the SPA inhibitor design strategy, exhibiting binding affinities ranging from substrate-like to exceeding that of the substrate by 100-fold. This rational approach for designing inhibitors of racemases and epimerases having the appropriate active-site architectures is a useful strategy for furnishing compounds for drug development.

Bacterial mandelic acid degradation pathway and its application in biotechnology.

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.

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.

Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery.

o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N-B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave Ki and Ki* values of 5.1 ± 1.8 and 0.26 ± 0.08 μM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by ∼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH3-treated enzyme-inhibitor complex did not yield evidence of an iminoboronate adduct. 11B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp3-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an Nζ-B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when o-carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.

Polymersomes as Nanoreactors Enabling the Application of Solvent-Sensitive Enzymes in Different Biphasic Reaction Setups.

There is an increasing interest in biocatalysis to perform chemical reactions in biphasic systems, consisting of an aqueous phase and a water-immiscible organic solvent or ionic liquid. In most cases, the hydrophobic phase is used as reservoir for poorly water-soluble substrates or for in situ product removal. However, many enzymes are solvent-sensitive and cannot be used in such systems. In this study, the solvent-sensitive enzyme mandelate racemase is exemplarily protected from the organic phase by its entrapment in (crosslinked) polymersomes. The covalent crosslinking of the individual chains of the block copolymer poly(2-methyloxazoline)15 -poly(dimethylsiloxane)68 -poly(2-methyloxazoline)15 via terminal methacrylates leads to enhanced membrane stability. This effect is especially pronounced for long-time incubation in the presence of organic solvents and ionic liquids. By using a gentle polymerization initiator at its minimal necessary concentration, the prior encapsulated enzymes remain intact during crosslinking. Although the insertion of natural channel proteins into the membrane improves the mass transport into the vesicles, it is non-essential. Mandelate racemase in (crosslinked) polymersomes remains active in different highly dispersed biphasic systems for more than 24h. The free enzyme, on the other hand, gets completely inactivated within 1h, thus illustrating the potential of polymersomes as nanoreactors in biphasic reaction setups.

Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center.

Boronic acids have been successfully employed as inhibitors of hydrolytic enzymes. Typically, an enzymatic nucleophile catalyzing hydrolysis adds to the electrophilic boron atom forming a tetrahedral species that mimics the intermediate(s)/transition state(s) for the hydrolysis reaction. We show that para-substituted phenylboronic acids (PBAs) are potent competitive inhibitors of mandelate racemase (MR), an enzyme that catalyzes a 1,1-proton transfer rather than a hydrolysis reaction. The Ki value for PBA was 1.8 ± 0.1 μM, and p-Cl-PBA exhibited the most potent inhibition (Ki = 81 ± 4 nM), exceeding the binding affinity of the substrate by ∼4 orders of magnitude. Isothermal titration calorimetric studies with the wild-type, K166M, and H297N MR variants indicated that, of the two Brønsted acid-base catalysts Lys 166 and His 297, the former made the greater contribution to inhibitor binding. The X-ray crystal structure of the MR·PBA complex revealed the presence of multiple H-bonds between the boronic acid hydroxyl groups and the side chains of active site residues, as well as formation of a His 297 Nε2-B dative bond. The dramatic upfield change in chemical shift of 27.2 ppm in the solution-phase 11B nuclear magnetic resonance spectrum accompanying binding of PBA by MR was consistent with an sp3-hybridized boron, which was also supported by density-functional theory calculations. These unprecedented findings suggest that, beyond substituting boron at carbon centers participating in hydrolysis reactions, substitution of boron at the acidic carbon center of a substrate furnishes a new approach for generating inhibitors of enzymes catalyzing the deprotonation of carbon acid substrates.

High level and enantioselective production of L-phenylglycine from racemic mandelic acid by engineered Escherichia coli using response surface methodology

L-Phenylglycine (L-PHG) is a member of unnatural amino acids, and becoming more and more important as intermediate for pharmaceuticals, food additives and agrochemicals. However, the existing synthetic methods for L-PHG mainly rely on toxic cyanide chemistry and multistep processes. To provide green, safe and high enantioselective alternatives, we envisaged cascade biocatalysis for the one-pot synthesis of L-PHG from racemic mandelic acid. A engineered E. coli strain was established to co-express mandelate racemase, D-mandelate dehydrogenase and L-leucine dehydrogenase and catalyze a 3-step reaction in one pot, enantioselectively transforming racemic mandelic acid to give L-PHG (e.e. >99 %). After the conditions for biosynthesis of L-PHG optimized by response surface methodology, the yield and space-time yield of L-PHG can reach 87.89 % and 79.70 g·L−1·d−1, which was obviously improved. The high-yielding and enantioselective synthetic methods use cheap and green reagents, and E. coli whole-cell catalysts, thus providing green and useful alternative methods for manufacturing L-PHG.

Attempts to develop an enzyme converting DHIV to KIV.

Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.

Altering the Y137-K164-K166 triad of mandelate racemase and its effect on the observed pKa of the Brønsted base catalysts

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate using a two-base mechanism with Lys 166 acting as the Brønsted base to abstract the α-proton from (S)-mandelate. The resulting intermediate is subsequently re-protonated by the conjugate acid of His 297 to yield (R)-mandelate. The roles of these amino acids are reversed when (R)-mandelate is the substrate. The side chains of Tyr 137, Lys 164, and Lys 166 form a H-bonding network and the proximity of the two ε-NH3+ groups is believed to lower the pKa of Lys 166. We used site-directed mutagenesis, kinetics, and pH-rate studies to explore the roles of Lys 164 (K164 C/M) and Tyr 137 (Y137 L/F/S/T) in catalysis. The efficiency (kcat/Km) was reduced ∼3.5 × 105-fold for K164C MR, relative to wild-type MR, indicating a major role for this residue in catalysis. The efficiency of Y137F MR, however, was reduced only 25–30-fold. pH-Rate profiles (log kcat vs. pH) revealed that substitution of Tyr 137 by Phe increased the kinetic pKa of Lys 166 from 5.88 ± 0.02 to 7.3 ± 0.2. Hence, Tyr 137 plays an important role in facilitating the reduction of the pKa of the Brønsted base Lys 166 by ∼1.4 units. Interestingly, the Phe substitution also increased the kinetic pKa of His 297 from 5.97 ± 0.04 to 7.1 ± 0.1. Thus, the Tyr 137-Lys 164-Lys 166 H-bonding network plays a broader role in modulating the pKa of catalytic residues by influencing the electrostatic character of the entire active site, not only by decreasing the observed pKa value of Lys 166, but also by decreasing the pKa of His 297 by 1.1 units.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis.

Phenylglyoxylic acid (PGA) are key building blocks and widely used to synthesize pharmaceutical intermediates or food additives. However, the existing synthetic methods for PGA generally involve toxic cyanide and complex processes. To explore an alternative method for PGA biosynthesis, we envisaged cascade biocatalysis for the one-pot synthesis of PGA from racemic mandelic acid. A novel mandelate racemase named ArMR showing higher expression level (216.9 U·mL-1 fermentation liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of mandelate racemase, d-mandelate dehydrogenase and l-lactate dehydrogenase, and transform racemic mandelic acid to PGA. Among them, the recombinant E. coli TCD 04, engineered to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic acid (100 mM) to PGA with 98% conversion. Taken together, we provide a green approach for one-pot biosynthesis of PGA from racemic mandelic acid.

Catalytic properties of the metal ion variants of mandelate racemase reveal alterations in the apparent electrophilicity of the metal cofactor.

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg2+, to catalyse the interconversion of the enantiomers of mandelate. Although the active site Mg2+ may be replaced by Mn2+, Co2+, or Ni2+, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters Kappm, kappcat, and (kcat/Km)app for the substrates (R)- and (S)-mandelate, and the alternative substrate (S)-trifluorolactate. Viscosity variation experiments with Mn2+-MR showed that the metal ion plays a role in the uniform binding of the transition states for enzyme-substrate association, the chemical step, and enzyme-product dissociation. Surprisingly, the competitive inhibition constants (Ki) for inhibition of each metalloenzyme variant by benzohydroxamate did not vary significantly with the identity of the metal ion unlike the marked variation of the stability constants (K1) observed for M2+·BzH complex formation in solution. A similar trend was observed for the inhibition of the metalloenzyme variants by F-, except for Mg2+-MR, which bound F- tighter than would be predicted based on the stability constants for formation of M2+·F- complexes in solution. Thus, the enzyme modifies the enatic state of the bound metal ion cofactor so that the apparent electrophilicity of Mg2+ is enhanced, while that of Ni2+ is attenuated, resulting in a levelling effect relative to the trends observed for the free metals in solution.

Genome mining and characterization of a new mandelate racemase

Racemases have been applied for the synthesis of enantiomerically pure compounds through the deracemization methods. Mandelate racemase from Pseudomonas putida was the only enzyme that catalyzes the interconversion of mandelate enantiomers. Using genome mining approaches, we identified 9 mandelate racemases (MRs). A novel racemase named ArMR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. ArMR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of ArMR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. KM parameter of ArMR towards (R)- and (S)-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding kcat value was 410 s⁻¹ and 218 s⁻¹. In addition, KM of ArMR towards (R)- and (S)-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding kcat value 0.22 s⁻¹ and 0.23 s⁻¹, respectively. Meanwhile, Mg²⁺ and Mn²⁺ could activate the enzyme whereas Zn²⁺ inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.

A platform for chemical modification of mandelate racemase: characterization of the C92S/C264S and γ-thialysine 166 variants.

Mandelate racemase (MR) serves as a paradigm for our understanding of enzyme-catalyzed deprotonation of a carbon acid substrate. To facilitate structure-function studies on MR using non-natural amino acid substitutions, we engineered the Cys92Ser/Cys264Ser variant (dmMR) as a platform for introducing Cys residues at specific locations for subsequent covalent modification. While the highly reactive thiol of Cys furnishes a site for chemical modification, site-specificity requires that other Cys residues be non-reactive or replaced by a non-reactive amino acid, especially if chemical modification is conducted under denaturing conditions. The catalytic efficiency of dmMR is reduced only ~2-fold relative to wild-type MR, making dmMR a viable platform for the site-specific introduction of Cys. As an example, the inactive Lys166Cys variant of dmMR was treated with ethylenimine under denaturing conditions to replace the Brønsted acid-base catalyst Lys 166 with the non-natural amino acid γ-thialysine. Comparison of the pH-activity profiles of dmMR and the active γ-thialysine variant revealed a reduction in the pKa for the side chain amino group of ~0.4 units for the latter variant. Unlike wild-type MR for which diffusion is partially rate-limiting, dmMR and the γ-thialysine variant showed no dependence on the solvent viscosity suggesting that the chemical step is fully rate-limiting.

Molecular dynamics perspective on the thermal stability of mandelate racemase

Mandelate racemase from Pseudomonas putida is a promising candidate for the dynamic kinetic resolution of α-hydroxy carboxylic acids. In the present study, the thermal stability of mandelate racemase was investigated through molecular dynamics simulations in the temperature range of 303–363 K, which can guide the design of mandelate racemase with higher stability. The basic features such as radius of gyration, surface accessibility, and secondary structure content suggested the instability of mandelate racemase at high temperatures. With increase in temperature, α-helix content reduced significantly, especially the α-helices exposed to the environment. At the simulation time scale considered, intra-protein hydrogen bonds, hydrogen bonds between protein and water decreased at 363 K, while the number of salt-bridges increased. The long-distance networks remarkably changed at 363 K. A considerable number of long-lived (percentage existence time higher than 90%) hydrogen bonds and Cα contacts were lost. Root mean square fluctuation analysis revealed regions with high fluctuation, which should be helpful in the reengineering of mandelate racemase for enhanced thermal stability.

Crystal structure analysis of 3,6-anhydro-l-galactonate cycloisomerase suggests emergence of novel substrate specificity in the enolase superfamily

3,6-Anydro-l-galatonate cycloisomerase (ACI) catalyzes the cycloisomerization of a 3,6-anhydro-l-galactonic acid known as a novel metabolite in agarolytic bacteria. Here, we present 3-D structures of ACI from Vibrio sp. strain EJY3 (VejACI) in native and mutant forms at 2.2 Å and 2.6 Å resolutions, respectively. The enzyme belongs to the mandelate racemase subgroup of the enolase superfamily catalyzing common β-elimination reactions by α-carbon deprotonation of substrates. The structure of VejACI revealed a notable 20s loop region in the capping domain, which can be a highly conserved structural motif in ACI homologs of agar metabolism. By comparing mutant (mVejAC/H300 N) and native VejACI structures, we identified a conformational change of Ile142 in VejACI that causes spatial expansion in the binding pocket. These observations imply that Ile142 and the 20s loop play important roles in enzymatic reactivity and substrate specificity. The structural phylogenetic analysis of the enolase superfamily including ACIs revealed sequential, structural, and functional relationships related to the emergence of novel substrate specificity.