Racemization of undesired enantiomers: Immobilization of mandelate racemase and application in a fixed bed reactor

Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high‐yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase. Immobilization of crude mandelate racemase via covalent attachment was optimized for two supports: Eupergit^® CM and CNBr‐activated Sepharose 4 Fast Flow. To allow coupling of enzymatic reaction with enantioselective chromatography, a mobile phase composition compatible with both processes was used in enzymatic reactor. Kinetic parameters obtained analyzing experiments carried out in a batch reactor could be successfully used to predict fixed‐bed reactor performance. The applicability of the immobilized enzyme and the determined kinetic parameters were validated in transient experiments recording responses to pulse injections of R‐mandelic acid. The approach investigated can be used for futher design and optimization of high yield combined resolution processes. The characterized fixed‐bed enzymatic reactor can be integrated e.g. with chromatographic single‐ or multicolumn steps in various configurations.     

Inhibition of mandelate racemase by alpha-fluorobenzylphosphonates

Mandelate racemase catalyzes the interconversion of the enantiomers of mandelic acid. The enzyme binds the intermediate analogues (R)- and (S)-alpha-fluorobenzylphosphonate, and alpha,alpha-difluorobenzylphosphonate with 100-2500 times less affinity than it exhibits for (R,S)-alpha-hydroxybenzylphosphonate at pH 7.5. This apparent low affinity, relative to that of alpha-hydroxybenzylphosphonate, arises from the altered pKa values of the alpha-fluorobenzylphosphonates. For example, (S)-alpha-fluorobenzylphosphonate is bound with the same affinity as the substrate at pH 7.5, but this affinity is increased approximately 6-fold at pH 6.3.     

Hydrophobic nature of the active site of mandelate racemase

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol. We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to evaluate the contribution of the hydrophobic cavity within the enzyme's active site to ligand binding. Free energy changes accompanying binding of glycolate derivatives correlated well with the hydrophobic substituent constant pi and the van der Waals surface areas of the ligands. The observed dependence of the apparent binding free energy on surface area of the ligand was -30 +/- 5 cal mol(-1) A(-2) at 25 degrees C. Free energy changes accompanying binding of hydroxamate derivatives also correlated well with pi values and the van der Waals surface areas of the ligands, giving a slightly greater free energy dependence equal to -41 +/- 3 cal mol(-1) A(-2) at 25 degrees C. Surprisingly, mandelate racemase exhibited a binding affinity for the intermediate analogue benzohydroxamate that was 2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions. This suggests that there are additional specific interactions that stabilize the altered substrate in the transition state. Mandelate racemase was competitively inhibited by (R,S)-1-naphthylglycolate (apparent K(i) = 1.9 +/- 0.1 mM) and (R,S)-2-naphthylglycolate (apparent K(i) = 0.52 +/- 0.03 mM), demonstrating the plasticity of the hydrophobic pocket. Both (R)- (K(m) = 0.46 +/- 0.06 mM, k(cat) = 33 +/- 1 s(-1)) and (S)-2-naphthylglycolate (K(m) = 0.41 +/- 0.03 mM, k(cat) = 25 +/- 1 s(-1)) were shown to be alternative substrates for mandelate racemase. These kinetic results demonstrate that no major steric restrictions are imposed on the binding of this bulkier substrate in the ground state but that steric factors appear to impair transition state/intermediate stabilization. 2-Naphthohydroxamate was identified as a competitive inhibitor of mandelate racemase, binding with an affinity (K(i) = 57 +/- 18 microM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the approximately 10-fold reduction in the value of k(cat)/K(m) for the racemization of 2-naphthylglycolate. These findings indicate that, for mandelate racemase, steric constraints within the hydrophobic cavity of the enzyme-intermediate complex are more stringent than those in the enzyme-substrate complex.     

Kinetics and thermodynamics of mandelate racemase catalysis

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutant enzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions. The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction. From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction [Bearne, S. L., and Wolfenden, R. (1997) Biochemistry 36, 1646-1656], we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/mol, TDeltaS = -4.8 +/- 0.8 kcal/mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/mol, TDeltaS# = +2.0 +/- 0.1 kcal/mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.     

Some properties of mandelate racemase from Pseudomonas fluorescens

1. l-Mandelate dehydrogenase and mandelate racemase were partially purified from extracts of Pseudomonas fluorescens A-312 grown in media containing d-mandelate. 2. The activity of mandelate racemase, but not that of l-mandelate dehydrogenase, is greatly stimulated by Mg(2+), Mn(2+), Co(2+) and, though less effectively, by Ni(2+). Other metal ions are inactive or inhibitory. 3. Racemase activity is inhibited by phosphate, fluoride, pyrophosphate and EDTA. The inhibitions by pyrophosphate and EDTA are competitive with respect to the metal ion activator; those by phosphate and fluoride are competitive with respect to the substrate. 4. The addition of Mg(2+) diminishes the Michaelis constant of racemase. 5. The pH optimum of the racemase is at 7.8. The pH-activity curve of the dehydrogenase complex of enzymes has two peaks, at 7.0 and 8.2. 6. The enzymic racemization of d-mandelate is initially faster than that of l-mandelate. 7. The rates of oxidation of related substrates, catalysed by l-mandelate dehydrogenase, are in the decreasing order: l-p-hydroxymandelate; l-3,4-dihydroxymandelate; l-4-hydroxy-3-methoxymandelate. The racemase is active towards d-p-hydroxymandelate but inactive towards d-3,4-dihydroxymandelate and d-4-hydroxy-3-methoxymandelate. Since 4-hydroxy-3-methoxymandelate, and presumably also 3,4-dihydroxymandelate, arising from the metabolism of catechol-amines, have the d-configuration, the enzymes studied cannot be utilized for estimation of the last two acids in urine.     

Preliminary x-ray data on crystals of mandelate racemase

The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.     

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.     

新型扁桃酸消旋酶的基因挖掘和性能表征

消旋酶是实现手性化合物去消旋化制备光学纯化学品的重要工具,来源于恶臭假单胞菌的扁桃酸消旋酶(MR),是目前唯一可以催化两种构型扁桃酸互相转换的消旋酶。通过基因组数据挖掘获得了9个新的扁桃酸消旋酶基因及活性蛋白,其中来源于放射性土壤杆菌Agrobacterium radiobacter的Ar MR酶对扁桃酸和邻氯扁桃酸具有较高的催化活力,而且该酶的异源表达水平也较理想。ArMR催化扁桃酸消旋反应的最适温度为50℃,最适pH为7.8。该酶在30℃、40℃和50℃下的半衰期分别为70.7、7.2、0.17 h。ArMR对(R)-和(S)-扁桃酸的K_M值分别为1.44 mmol/L和0.81 mmol/L,k_(cat)值分别为410 s~(–1)和218 s~(–1);对(R)-和(S)-邻氯扁桃酸的KM值分别为6.48 mmol/L和6.37 mmol/L,而k_(cat)值为0.22 s~(–1)和0.23 s~(–1)。Mg~(2+)和Mn~(2+)对该酶的活力有促进作用,而Zn~(2+)使其完全失活。新型扁桃酸消旋酶的发现和表征为今后此类酶的深入研究和开发提供了更多资源和数据参考。     

Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-A resolution: identification of the active site and possible catalytic residues

The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.     

Purification of recombinant mandelate racemase: Improved catalytic activity

Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg²⁺-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pK_a values. Purification of recombinant MR as a fusion protein with an N-terminal hexahistidine tag using immobilized-nickel ion affinity chromatography and elution with a linear gradient of EDTA revealed three enzyme species (mrI, mrII, and mrIII). While mrIII was catalytically inactive, both mrI and mrII catalyzed the racemization of (S)-mandelate with turnover numbers (k_cat) of 190 ± 22 and 940 ± 24 s^?1, respectively. Circular dichroism analysis suggested that mrIII was a partially unfolded or misfolded form of the enzyme. Replacement of the N-terminal hexahistidine tag by a StrepII-tag appeared to ameliorate the folding problem yielding a single enzyme species with a turnover number of 1124 ± 43 s^?1. The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (k_cat = 472 ± 37 s^?1). These results highlight a potential problem that may be encountered when producing fusion enzymes bearing a polyhistidine tag: soluble, active enzyme may be obtained but care must be taken to ensure that it is free of minor misfolded forms that can alter the apparent activity of the enzyme.     

Substrate spectrum of mandelate racemase: Part 2. (Hetero)-aryl-substituted mandelate derivatives and modulation of activity

Efficient enzymatic racemization of 2-hydroxy-2-heteroaryl-acetic acid derivatives by mandelate racemase under mild conditions is reported for the first time. (i) Steric limitations for aryl-substituted mandelate derivatives were elucidated to be particularly striking for o-substituents, whereas m- and p-analogues were freely accepted, as well as heteroaryl- and naphthyl-analogs. (ii) The electronic character of substituents was found to play an important role: whereas electron-withdrawing substituents dramatically enhanced the racemization rates, electron-donating analogs caused a depletion. This effect could be ascribed to an α-carbanion-stabilization in accordance with the known enzyme mechanism. The latter was modeled by comparison of gas phase deprotonation energies as a useful parameter to describe resonance stabilization. The calculated data nicely correlate with the experimentally observed activities for a specific substrate as long as other parameters, such as steric restrictions, are absent or play a minor role.     

Intermediate analogue inhibitors of mandelate racemase: N-Hydroxyformanilide and cupferron

Mandelate racemase (MR) catalyzes the 1,1-proton transfer that interconverts the enantiomers of mandelate. The transition state/intermediate analogues N-hydroxyformanilide (K(i)=2.79+/-0.19 microM) and cupferron (K(i)=2.67+/-0.09 microM) are identified as potent competitive inhibitors of MR. The pH-pK(i) profile indicates that MR can bind either the protonated or deprotonated forms of N-hydroxyformanilide, with a 10-fold greater affinity for the latter form.     

Inhibition of mandelate racemase by the substrate-intermediate-product analogue 1,1-diphenyl-1-hydroxymethylphosphonate

Mandelate racemase has been studied as a paradigm for enzyme-catalyzed abstraction of a proton from carbon acids with relatively high pKa values. 1,1-Diphenyl-1-hydroxymethylphosphonate is a substrate-intermediate-product analogue and is a modest competitive inhibitor of the enzyme (Ki=1.41+/-0.09 mM), suggesting that simultaneous binding of the two phenyl groups obviates mimicry of the aci-carboxylate function of the intermediate by the phosphonate group.     

Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.     

Structure of mandelate racemase with bound intermediate analogues benzohydroxamate and cupferron

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-? resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and Cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, Cupferron, and the ground state analogue (S)-atrolactate reveal that the para carbon of the substrate phenyl ring moves by 0.8-1.2 ? between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to that of MR with bound (S)-atrolactate, the intermediate-Mg(2+) distance becomes shorter, suggesting a tighter complex with the catalytic Mg(2+). In addition, Tyr 54 moves closer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle.     

Directed evolution of mandelate racemase by a novel high-throughput screening method

Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated k _cat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.

     

Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism

The fate of the alpha-hydrogen of mandelate in the reaction catalyzed by mandelate racemase has been investigated by a mass spectroscopic method. The method entails the incubation of (R)- or (S)-[alpha-1H]mandelate in buffered D2O to a low extent of turnover (about 5-8%), esterification of the resulting mixture of mandelates with diazomethane, derivatization of the methyl esters with a chiral derivatizing agent, and quantitation of the isotope content of the alpha-hydrogen of both substrate and product by gas chromatography/mass spectrometric analysis. No significant substrate-derived alpha-protium was found in the product for racemization in either direction. In addition, in the (R) to (S) direction almost no exchange (less than or equal to 0.4%) of the alpha-hydrogen in the remaining (R) substrate pool occurred, but in the (S) to (R) direction 3.5-5.1% exchange of the alpha-hydrogen in the remaining substrate (after 5.1-7.2% net turnover) was found. Qualitatively similar results were obtained in the (S) to (R) direction in H2O when (S)-[alpha-2H]mandelate was used as substrate. In other experiments, an overshoot in the progress curve was observed when the racemization of either enantiomer of [alpha-1H]mandelate in D2O was monitored by following the change in ellipticity of the reaction mixture; the magnitude of the overshoot was greater in the (R) to (S) than in the (S) to (R) direction. All of the available data indicate that the reaction catalyzed by mandelate racemase proceeds by a two-base mechanism, in contrast to earlier proposals.