Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Neurobiology through the looking-glass: D-serine as a new glial-derived transmitter

D-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of D-amino acids in mammals challenge classic concepts in biology in which only L-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous D-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free D-serine levels in the brain are about one third of L-serine values and its extracellular concentration is higher than many common L-amino acids. D-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous D-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. The enzyme requires pyridoxal 5'-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous D-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.     

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.     

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.     

Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The Vmax/Km values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.     

The N-methyl D-aspartate receptor glycine site and D-serine metabolism: an evolutionary perspective

The N-methyl D-aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous d-serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D-serine in brain. Sequence alignments of the glycine-binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D-serine in brain is due to a pyridoxal-5'-phosphate (B(6))-requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B(6) enzymes, along with the known occurrences of serine racemases in animals, point to D-serine synthesis arising around the divergence time of arthropods. D-Serine catabolism occurs via the ancient peroxisomal enzyme d-amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D-serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics.     

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.     

D-Amino acid metabolism in mammals: biosynthesis, degradation and analytical aspects of the metabolic study

It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of D-amino acids are present in mammals. The most abundant D-amino acids are D-serine and D-aspartate. D-Serine, which is synthesized by serine racemase and is degraded by D-amino-acid oxidase, is present in the brain and modulates neurotransmission. D-Aspartate, which is synthesized by aspartate racemase and degraded by D-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. D-Serine and D-aspartate bind to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these D-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and D-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on D-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and D-amino-acid oxidases.     

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.     

D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration

The mammalian brain contains unusually high levels of D-serine, a D-amino acid previously thought to be restricted to some bacteria and insects. In the last few years, studies from several groups have demonstrated that D-serine is a physiological co-agonist of the N-methyl D-aspartate (NMDA) type of glutamate receptor -- a key excitatory neurotransmitter receptor in the brain. D-Serine binds with high affinity to a co-agonist site at the NMDA receptors and, along with glutamate, mediates several important physiological and pathological processes, including NMDA receptor transmission, synaptic plasticity and neurotoxicity. In recent years, biosynthetic, degradative and release pathways for D-serine have been identified, indicating that D-serine may function as a transmitter. At first, D-serine was described in astrocytes, a class of glial cells that ensheathes neurons and release several transmitters that modulate neurotransmission. This led to the notion that D-serine is a glia-derived transmitter (or gliotransmitter). However, recent data indicate that serine racemase, the D-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that D-serine also has a neuronal origin. We now review these findings, focusing on recent questions regarding the roles of glia versus neurons in d-serine signaling.     

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.     

Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP

Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.     

D-amino acids in the brain: structure and function of pyridoxal phosphate-dependent amino acid racemases

D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best-studied enzyme among the PLP-dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.     

Serine racemases from barley, Hordeum vulgare L., and other plant species represent a distinct eukaryotic group: gene cloning and recombinant protein characterization

Several d-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid residues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved pyridoxal 5-phosphate (PLP)-binding lysine and PLP-interacting amino acid residues. The purified gene product catalyzed not only racemization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca(2+), Mg(2+), or Mn(2+), but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine racemase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types.     

Serine racemase and the serine shuttle between neurons and astrocytes

d-Serine is a brain-enriched d-amino acid that works as a transmitter-like molecule by physiologically activating NMDA receptors. Synthesis of d-serine is carried out by serine racemase (SR), a pyridoxal 5'-phosphate-dependent enzyme. In addition to carry out racemization, SR α,β-eliminates water from l- or d-serine, generating pyruvate and NH(4)(+). Here I review the main mechanisms regulating SR activity and d-serine dynamics in the brain. I propose a role for SR in a novel form of astrocyte-neuron communication-the "serine shuttle", whereby astrocytes synthesize and export l-serine required for the synthesis of d-serine by the predominantly neuronal SR. d-Serine synthesized and released by neurons can be further taken up by astrocytes for storage and activity-dependent release. I discuss how SR α,β-elimination with d-serine itself may limit the achievable intracellular d-serine concentration, providing a mechanistic rationale on why neurons do not store as much d-serine as astrocytes. The higher content of d-serine in astrocytes appears to be related to increased d-serine stability, for their low SR expression will prevent substantial d-serine metabolism via α,β-elimination. SR and the serine shuttle pathway are therapeutic targets in neurodegenerative diseases in which NMDA receptor dysfunction plays pathological roles. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

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.     

D-Serine as a putative glial neurotransmitter

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the 'glycine' site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.     

Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.