Mechanism of C-3 hydrogen exchange and the elimination of ammonia in the 3-methylaspartate ammonia-lyase reaction.

The enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) catalyzes the exchange of the C-3 hydrogen of the substrate, (2S,3S)-3-methylaspartic acid, with solvent hydrogen. The mechanism of the exchange reaction was probed using (2S,3S)-3-methylaspartic acid and its C-3-deuteriated isotopomer. Incubations conducted in tritiated water allowed the rate of protium or deuterium wash-out from the substrates to be measured as tritium wash-in. The primary deuterium isotope effects for the exchange under essentially Vmax conditions ( [S] much greater than Km) were 1.6, 1.5, and 1.5 at pH 9.0, 7.6, and 6.5. The deamination reaction, measured spectrophotometrically on the same incubations, showed isotope effects of 1.7, 1.6, and 1.4 at pH 9.0, 7.6, and 6.5, in agreement with the values of DV and D(V/K) reported previously [Botting, N.P., Akhtar, M., Cohen, M.A., & Gani, D. (1988) Biochemistry 27, 2956-2959]. The ratio of the rate of exchange to the rate of deamination, however, varied widely with pH. Together with the identical values of the primary isotope effects for the two reactions, this result indicates that the partition between reaction pathways occurs after the slowest steps in the common part of the reaction coordinate pathway, almost certainly after the cleavage of the C-N bond at the level of the enzyme-ammonia-mesaconic acid complex, and not at the putative carbanion level as was previously suggested. The enzyme requires both K+ and Mg2+ ions for activity, although ammonium ion is also able to bind in the K+ site and act as an activator. Variation of the metal ion concentration alters the magnitude of the primary deuterium isotope effects. The variation of potassium ion concentration causes the most marked changes: at 1.6 mM K+, DV and D(V/K) are 1.7, whereas at 50 mM K+, DV and D(V/K) are reduced to 1.0. The isotope effects are also reduced at low K+ concentration due to the emergence of a slow-acting high K+ affinity monopotassium form of the enzyme. The binding order and role of the metal ion cofactors and their influence in determining the formal mechanism of the reaction is discussed, and the failure of previous workers to observe primary deuterium isotope effects for the deamination process is explained. The product desorption order was tested by product inhibition, alternative product inhibition, and isotope exchange experiments. Ammonia and mesaconic acid debind in a random fashion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: identification of an essential cysteine residue.

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3.     

The 3-methylaspartase reaction probed using 2H- and 15N-isotope effects for three substrates: a flip from a concerted to a carbocationic amino-enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S.

The mechanisms of the elimination of ammonia from (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid, catalysed by the enzyme L-threo-3-methylaspartase ammonia-lyase (EC 4.3.1.2) have been probed using 15N-isotope effects. The 15N-isotope effects for V/K for both (2S,3S)-3-methylaspartic acid and aspartic acid are 1.0246 +/- 0.0013 and 1.0390 +/- 0.0031, respectively. The natural substrate, (2S,3S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C(beta)-H and C(alpha)-N bonds are cleaved in the same transition state. (2S)-Aspartic acid appears to follow the same mechanistic pathway, but deprotonation of the conjugate acid of the base for C-3 is kinetically important and influences the extent of 15N-fractionation. (2S,3R)-3-Methylaspartic acid is deaminated via a stepwise carbocationic mechanism. Here we elaborate on the proposed model for the mechanism of methylaspartase and propose that a change in stereochemistry of the substrate induces a change in the mechanism of ammonia elimination.     

3-Methylaspartate ammonia-lyase from a facultative anaerobe,strain YG-1002

3-Methylaspartase was purified 24-fold and crystallized from the crude extract of the cells of a facultative anaerobic bacterium from soil, strain YG-1002. The molecular mass of the native enzyme was about 84 kDa and that of the subunit was about 42 kDa. The pH optimum for the deamination reaction of (2S, 3S)-3-methylaspartic acid and those for the amination reaction of mesaconic acid were 9.7 and 8.5; its optimum temperature was 50°C. The enzyme was stable at pH 5.5–11.0 and up to 50°C. The enzyme required both divalent and monovalent cations such as Mg²⁺ and K⁺. The enzyme was inhibited by sulfhydryl reagents, metal-chelating reagents and some divalent cations. The enzyme catalyzed the reversible amination/deamination reactions between several 3-substituted (S)-aspartic acids and their corresponding fumaric acid derivatives. The enzyme preferentially acted on (2S, 3S)-3-methylaspartic acid and mesaconic acid in the deamination and the amination reactions respectively. The enzyme showed high similarities in several enzymological properties and N-terminal amino acid sequence with 3-methylaspartase from an obligate anaerobic bacteriumClostridium tetanomorphum.     

Kinetic mechanism and location of rate-determining steps for aspartase from Hafnia alvei

Coupled spectrophotometric assays that monitor the formation of fumarate and ammonia in the direction of aspartate deamination and aspartate in the direction of fumarate amination were used to collect initial velocity data for the aspartase reaction. Data are consistent with rapid equilibrium ordered addition of Mg2+ prior to aspartate but completely random release of Mg2+, NH4+, or fumarate. In addition to Mg2+, Mn2+ can also be used as a divalent metal with Vmax 80% and a Kaspartate 3.5-fold lower than when Mg2+ is used. Monovalent cations such as Li+, K+, Cs+, and Rb+ are competitive vs. either aspartate or NH4+ but noncompetitive vs. fumarate. A primary deuterium isotope effect of about 1 on both V and V/Kaspartate is obtained with (3R)-L-aspartate-3-d, while a primary 15N isotope effect on V/Kaspartate of 1.0239 +/- 0.0014 is obtained in the direction of aspartate deamination. A secondary isotope effect on V of 1.13 +/- 0.04 is obtained with L-aspartate-2-d. In addition, a secondary isotope effect of 0.81 +/- 0.05 on V is obtained with fumarate-d2, while a value of 1.18 +/- 0.05 on V is obtained by using (2S,3S)-L-aspartate-2,3-d2. These data are interpreted in terms of a two-step mechanism with an intermediate carbanion in which C-N bond cleavage limits the overall rate and the rate-limiting transition state is intermediate between the carbanion and fumarate.     

Isotopic probes of the argininosuccinate lyase reaction

The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotopic substitution at the reaction centers. A primary deuterium isotope effect of 1.0 on both V and V/K is obtained with (2S,3R)-argininosuccinate-3-d, while a primary 15N isotope effect on V/K of 0.9964 +/- 0.0003 is observed. The 15N isotope effect on the equilibrium constant is 1.018 +/- 0.001. The proton that is abstracted from C-3 of argininosuccinate is unable to exchange with the solvent from the enzyme-intermediate complex but is rapidly exchanged with solvent from the enzyme-fumarate-arginine complex. A deuterium solvent isotope effect of 2.0 is observed on the Vmax of the forward reaction. These and other data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the inverse 15N primary isotope effect and the solvent deuterium isotope effect suggest that protonation of the guanidino group and carbon-nitrogen bond cleavage of argininosuccinate are kinetically significant.     

Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein.

The gene encoding methylaspartase (EC 4.3.1.2) from Clostridium tetranomorphum has been cloned, sequenced, and expressed in Escherichia coli. The open reading frame (ORF) codes for a polypeptide of 413 amino acid residues (M(r) 45,539) of which seven are cysteine residues. The size of the ORF indicates that methylaspartase is a homodimer rather than an (AB)2 tetramer. The deduced primary structure of the protein shows no homology to enzymes that catalyze similar reactions or, indeed, any convincing homology with any other characterized protein. The recombinant protein is identical to the enzyme isolated directly from C. tetanomorphum as determined by several criteria. The enzyme is obtained in a highly active form (approximately 70% of the activity of the natural enzyme) and migrates as a single band (M(r) 49,000) in SDS-polyacrylamide gels. The kinetic parameters for the deamination of (2S,3S)-3-methylaspartic acid by the natural and recombinant proteins are very similar, and the proteins display identical potassium ion-dependent primary deuterium isotope effects for V and V/K when (2S,3S)-3-methylaspartic acid is employed as the substrate. In accord with the activity of the natural enzyme, the recombinant protein is able to catalyze the slow formation of (2S,3R)-3-methylaspartic acid, the L-erythro-epimer of the natural substrate, from mesaconic acid and ammonia. Earlier work in which the cysteine residues in the protein were labeled with N-ethylmaleimide had indicated that there were eight cysteine residues per protein monomer. One cysteine residue was protected by substrate. Here evidence is forwarded to suggest that the residue that was protected by the substrate is not a cysteine residue but the translation product of a serine codon. Kinetic data indicate that this serine residue may be modified in the active enzyme. The implications of these findings on the mechanism of catalysis are discussed within the context of a few emerging mode of action for methylaspartate ammonia-lyase.     

Use of an altered sugar-nucleotide to unmask the transition state for alpha(2-->6) sialyltransferase

Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.     

Substrate specificity of the 3-methylaspartate ammonia-lyase reaction: observation of differential relative reaction rates for substrate-product pairs

A range of substituted fumaric and aspartic acid substrates for the enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) have been synthesized and used to study the kinetics of the catalyzed reaction in both the forward (deamination) and reverse (conjugative amination) reaction directions. The rates of amination for all of the alpha, beta-unsaturated substrates studied (bearing substituents the size of an ethyl group or smaller) were similar under [s] much greater than KM conditions although KM values for the substrates varied by a factor of 25. The rates of deamination for the corresponding 3-substituted amino acid substrates varied widely with structure under [s] much greater than KM conditions, and thus for substrate-product pairs the ratio for V(forward)/V(reverse) also varied. These differential reaction rates indicate that there is a step in the deamination direction that is especially sensitive to the size of the 3-substituent of the substrate and that a relatively large group (methyl to ethyl in size) is required for binding in order to reduce the activation energy for this step. Given that it is proposed that the enzyme operates via an E1cb-type mechanism where C-N bond cleavage is rate limiting, it is likely that binding of the C-3 substituent of aspartic acid substrates affects the alignment of the nascent carbanion with the C-N bond for elimination.     

Nmr study of poly(aspartic acid). I. α- and β-Peptide bonds in poly(aspartic acid) prepared by thermal polycondensation

The structure of poly(aspartic acid) prepared by thermal polycondensation has been studied by means of nmr spectroscopy. The analysis of the ¹³C-nmr spectra of the polymer at various pH values and comparison with the spectrum of poly(α-L -aspartic acid) revealed that the polymer contained aspartic acid linked in α- and β-peptide bonds. The mole fraction of the β-peptide bonds has been found to be 0.8 ± 0.1. The significance of the results for the evolutionary theory of S. W. Fox is mentioned.     

Incorporation of (2S,3S) and (2S,3R) beta-methyl aspartic acid into RGD-containing peptides

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) beta-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors alpha(IIb)beta3 and alpha(v)beta3 and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) beta-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) beta-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics.     

Total synthesis and antifungal activity of (2S,3R)-2-aminododecan-3-ol

We report the total synthesis of (2S,3R)-2-aminododecan-3-ol has been achieved starting from commercially available 10-undecenoic acid. The key steps involved are Sharpless asymmetric epoxidation, Miyashita's boron-directed C-2 regioselective azidolysis, generated the asymmetric centers and in situ detosylation and reduction of azido tosylate. The antifungal activity of the synthesized (2S,3R)-2-aminododecan-3-ol was evaluated on several Candida strains and was comparable to miconazole, a standard drug.     

Structure of a novel cofactor containing N-(7-mercaptoheptanoyl)-O-3-phosphothreonine

The cofactor required in the methylcoenzyme M methylreductase reaction was shown to be a large molecule with an Mr of 1149.21 in the free acid form. The cofactor, named MRF for methyl reducing factor, was identified from analyses by fast atom bombardment mass spectrometry and 1H, 13C, and 31P NMR spectroscopy as uridine 5'-[N-(7-mercaptoheptanoyl)-O-3-phosphothreonine-P-yl(2-acetamido- 2-deoxy- beta-mannopyranuronosyl)(acid anhydride)]-(1----4)-O-2-acetamido-2-deoxy- alpha-glucopyranosyl diphosphate. MRF contains N-(7-mercaptoheptanoyl)threonine O-3-phosphate (HS-HTP) [No11, K. M., Rinehart, K. L., Tanner, R. S., & Wolfe, R. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242] and is linked to C-6 of 2-acetamido-2-deoxymannopyranuronic acid of the UDP-disaccharide through a carboxylic-phosphoric anhydride linkage. It is postulated that this bond is responsible for the instability of the molecule and its hydrolysis during isolation. Analyses of Eadie and Hofstee plots of the methylcoenzyme M methylreductase reaction indicate that MRF has a 6-fold lower Km(app) than HS-HTP and a 50% greater Vmax. This suggests that the UDP-disaccharide moiety may be of importance in the binding of MRF to the enzyme active site.     

Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata

Deuterium-labeled dihydroorotates bearing one, two, or three deuteriums at the pair of C4 and C5 positions have been synthesized in high isotopic and chiral purity and characterized by NMR and mass spectroscopy. These substrates have been used with the FMN-containing biosynthetic dihydroorotate oxidase from Crithidia fasciculata [Pascal, R., Trang, N., Cerami, A., & Walsh, C. (1983) Biochemistry 22, 171] to probe stereochemistry and mechanism. At pH 6.0 the (4RS)-[5,5-2H2]dihydroorotate shows a Vmax isotope effect (DV) of 2.83; since the (4S,5R)-[5-2H]dihydroorotate shows a DV of no more than 1.1, a secondary effect, the overall stereochemistry of desaturation is anti as previously reported for the degradative orotate reductase from Clostridium oroticum. The (4RS)-[4-2H]dihydroorotate shows a DV of 2.97, indicating removal of the C4-H is also partially rate limiting at pH 6.0. When trideuterio (4RS)-[4,5,5-2H3]dihydroorotate was tested, a DV of 8.0, a value close to the product of the separate isotope effects at the 4- and 5S-positions, was observed. At this pH then, both C-H cleavage steps are partly rate limiting in catalysis. Under anaerobic conditions without an electron acceptor the enzyme catalyzes the preferential exchange of the 5S hydrogen with solvent protons. The aggregate isotope effects on Vmax (DV) and on Vmax/Km [D(V/K)] are analyzed and suggest a stepwise rather than a concerted mechanism for this biosynthetic desaturation in pyrimidine biosynthesis.     

Crotonase-catalyzed beta-elimination is concerted: a double isotope effect study

Determining the sequence of bond cleavages, and consequently the nature of intermediates, in enzyme-catalyzed reactions is a major goal of mechanistic enzymology. When significant primary isotope effects on V/K are observed for two different bond cleavages, both bonds may be broken in the same transition state or they can reflect two different transition states that are of nearly identical energy and consequently both are partially rate limiting. For the crotonase-catalyzed dehydration of 3-hydroxybutyrylpantetheine, the primary D(V/K) and 18(V/K) are 1.60 and 1.053 [Bahnson, B. J., & Anderson, V. E. (1989) Biochemistry 28, 4173-4181], respectively. In this case, double isotope effects can discriminate between the two possibilities [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114; Belasco, J. G., Albery, W. J., & Knowles, J. R. (1983) J. Am. Chem. Soc. 105, 2475-2477]. The ratio of the alpha-secondary D(V/K) for the hydration of crotonylpantetheine catalyzed by crotonase in H2O and D2O has been determined to be 1.003 +/- 0.006. The invariance of the alpha-secondary effect where the chemical reaction is completely rate determining requires that both bond cleavages be concerted or that the substitution of 2H at the primary position not significantly alter the partitioning of a hypothetical carbanion. The observation of a solvent discrimination isotope effect determined from the relative incorporation of 2H from 50% D2O of 1.60 +/- 0.03, identical with the primary D(V/K), and the determination that the rate of exchange of the abstracted proton with solvent proceeds at less than 3% of the overall reaction rate also fail to provide evidence for a carbanion intermediate and are consistent with a concerted reaction. Identical primary D(V/K)s determined in H2O and D2O indicate that there is not a significant solvent isotope effect on C-O bond cleavage. The isotope ratios determined in these studies were performed by negative ion chemical ionization whole molecule mass spectrometry of the pentafluorobenzyl esters, a new method whose validity is established by comparison with previously determined kinetic and equilibrium isotope effects.     

Crystalline 3-methylaspartase from a facultative anaerobe, Escherichia coli strain YG1002

Abstract Crystalline 3-methylaspartase (EC 4.3.1.2) from Escherichia coli strain YG1002 that had been isolated from soil was characterized. The enzyme activity was induced when the organism was grown statically on medium containing ( S )-glutamic acid. Its molecular mass is about 84 kDa, and it may be composed of two identical subunits of 42 kDa. The enzyme requires both divalent and monovalent cations such as Mg²⁺ and K⁺, respectively. The enzyme catalyzes reversible amination-deamination between mesaconic acid and (2 S ,3 S )-methylaspartic acid, which is the best substrate.     

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor

β-methylaspartate ammonia-lyase, EC 4.3.1.2, (β-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b , respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)- 3a yielded the α,β-substituted β-lactone (3R,4R) 6 with a very high diastereoisomeric excess (>95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl β-(2R,3S)-3-methylmalate] 8 , prepared by an anionic ring opening polymerization, was highly isotactic as determined by ¹³C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7 , to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of β-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions. Chirality 10:727–733, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

NAD(+)-dependent (S)-specific secondary alcohol dehydrogenase involved in stereoinversion of 3-pentyn-2-ol catalyzed by Nocardia fusca AKU 2123

An NAD(+)-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K(m) and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 mumol/min/mg, and 0.33 mM and 130 mumol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K(m) and Vmax for 2-hexanone reduction were 2.5 mM and 260 mumol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.