Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum.

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.     

Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism

Pyrococcus horikoshii OT3 aspartate racemase (PhAspR) catalyzes the interconversion between L- and D-aspartate. The X-ray structure of PhAspR revealed a pseudo mirror-symmetric distribution of the residues around its active site, which is very reasonable for its chiral substrates, L-aspartate and D-aspartate. In this study, we have determined the crystal structure of an inactive mutant PhAspR complexed with a citric acid (Cit) at a resolution of 2.0 A. Cit contains the substrate analogue moieties of both L- and D-aspartate and exhibits a low competitive inhibition activity against PhAspR. In the structure, Cit binds to the catalytic site of PhAspR, which induced the conformational change to close the active site. The distance between the thiolates was estimated to be 7.4 A, representing a catalytic state and the substrate binding modes of PhAspR. Two conserved basic residues, Arg48 and Lys164, seem to be indispensable for PhAspR activity. Arg48 is thought to be responsible for recognizing carboxyl groups of the substrates L-/D-aspartates and stabilizing a reaction intermediate, and Lys164 is responsible for stabilizing a closed state structure. In this structure, the L-aspartate moiety of Cit is likely to take the substrate position of the PhAspR-substrate complex, which is very similar to that of Glutamate racemase. There is also another possibility that the two substrate analogue moieties of the bound Cit reflect the binding modes of both L- and D-aspartates. Based on the PhAspR-Cit complex structure, the reaction mechanism of aspartate racemase was elucidated.     

Purification and characterization of aspartate racemase from the bivalve mollusk Scapharca broughtonii

High concentrations of D-aspartate occur in blood shell Scapharca broughtonii (Mollusca) tissues. We purified aspartate racemase from the foot muscle of the bivalve to electrophoretic homogeneity. The molecular mass shown by sodium dodecyl sulfate polyacrylamide gel was 39 kDa, while that shown by gel filtration ranged from 51 to 63 kDa. Pyridoxal 5'-phosphate-dependency of the enzyme was demonstrated by its absorption spectrum as well as the effects of amino-oxyacetate and other reagents on the activity and spectrum. The enzyme is highly specific to aspartate and does not racemize L-alanine, L-serine and L-glutamate. It showed the highest activity at pH 8 both in the conversion of L- to D- and D- to L-aspartate, and the optimal temperature was 25 degrees C. V(max) and K(m) values for L-aspartate were 7.39 micromolmin(-1)mg(-1) and 60.4 mM and those for D-aspartate were 22.6 micromolmin(-1)mg(-1) and 159 mM, respectively.     

D-amino acids in the brain: the biochemistry of brain serine racemase

It has been recently established that in various brain regions D-serine, the product of serine racemase, occupies the so-called 'glycine site' within N-methyl D-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5' phosphate-containing enzyme that catalyzes the racemization of L-serine to D-serine. It has also been shown to catalyze the alpha,beta-elimination of water from L-serine or D-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5' phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein interacting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above.     

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.     

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.     

Occurrence of freed-aspartate and aspartate racemase in the blood shellScapharca broughtonii

Summary Substantial concentrations of D-aspartate were found in several tissues of Scapharca broughtonii together with approximately equal concentrations ofl-aspartate. The foot and mantle extracts also contained an aspartate racemase activity. The formation ofl-aspartate from the Denantiomer by the foot extract was apparently slower than the reverse reaction, and this unbalance seemed to be due to the presence of an enzyme activity which rapidly convertedl-aspartate tol-alanine. The possible role of D-aspartate in the anaerobiosis was discussed.     

Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.

Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.     

Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine racemase

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.     

Reaction mechanism and structure of the active site of proline racemase.

Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.     

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.     

Cloning and expression of the pyridoxal 5'-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme

D-aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.     

A novel pyridoxal 5'-phosphate-dependent amino acid racemase in the Aplysia californica central nervous system

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

Reaction mechanism of alanine racemase from Bacillus stearothermophilus: x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine

The crystal structures of alanine racemase bound with reaction intermediate analogs, N-(5'-phosphopyridoxyl)-L-alanine (PLP-L-Ala) and N-(5'-phosphopyridoxyl)-D-alanine (PLP-D-Ala), were determined at 2.0-A resolution with the crystallographic R factor of 17.2 for PLP-L-Ala and 16.9 for PLP-D-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5'-phosphate (PLP)-form enzyme; root mean square deviations at Calpha among the three structures were less than 0.28 A. The side chains of the amino acid residues around the PLP-L-Ala and PLP-D-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The alpha-hydrogen of the alanine moiety of PLP-L-Ala was located near the OH of Tyr(265)', whereas that of PLP-D-Ala was near the NZ of Lys(39). These support the previous findings that Tyr(265)' and Lys(39) are the catalytic bases removing alpha-hydrogen from L- and D-alanine, respectively. The prerequisite for this two-base mechanism is that the alpha-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys(39) and the OH of Tyr(265'); otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys(39) and Tyr(265)'. The results of molecular orbital calculation also support this mechanism.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.