Publications

• The structure of mammalian serine racemase: evidence for conformational changes upon inhibitor binding.

  Smith M.A., Mack V., Ebneth A., Moraes I., Felicetti B., Wood M., Schonfeld D., Mather O., Cesura A., Barker J.

  Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L-to D-serine and serine catabolism by alpha,bet ... >> More

  Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L-to D-serine and serine catabolism by alpha,beta-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of beta-family pyridoxal 5'-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate. << Less

  J. Biol. Chem. 285:12873-12881(2010) [PubMed] [EuropePMC]

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

  Foltyn V.N., Bendikov I., De Miranda J., Panizzutti R., Dumin E., Shleper M., Li P., Toney M.D., Kartvelishvily E., Wolosker H.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 280:1754-1763(2005) [PubMed] [EuropePMC]

• Modulating the function of human serine racemase and human serine dehydratase by protein engineering.

  Wang C.Y., Ku S.C., Lee C.C., Wang A.H.

  D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, res ... >> More

  D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, respectively. hSR and hSDH have significant sequence homology (28% identity) and are similar in their structural folds (root-mean-square deviation, 1.12 Å). Sequence alignment and structural comparison between hSR and hSDH reveal that S84 in hSR and A65 in hSDH play important roles in their respective enzyme activities. We surmise that exchange of these two amino acids by introducing S84A hSR and A65S hSDH mutants may result in switching their protein functions. To understand the modulating mechanism of the key residues, mutants S84A in hSR and A65S in hSDH were constructed to monitor the change of activities. The structure of A65S hSDH mutant was determined at 1.3 Å resolution (PDB 4H27), elucidating the role of this critical amino acid. Our study demonstrated S84A hSR mutant behaved like hSDH, whereas A65S hSDH mutant acquired an additional function of using D-serine as a substrate. << Less

  Protein Eng Des Sel 25:741-749(2012) [PubMed] [EuropePMC]

• Structural basis for the broad specificity of a new family of amino-acid racemases.

  Espaillat A., Carrasco-Lopez C., Bernardo-Garcia N., Pietrosemoli N., Otero L.H., Alvarez L., de Pedro M.A., Pazos F., Davis B.M., Waldor M.K., Hermoso J.A., Cava F.

  Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mu ... >> More

  Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members. << Less

  Acta Crystallogr. D 70:79-90(2014) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• Serine racemase with catalytically active lysinoalanyl residue.

  Yamauchi T., Goto M., Wu H.Y., Uo T., Yoshimura T., Mihara H., Kurihara T., Miyahara I., Hirotsu K., Esaki N.

  Serine racemase synthesizes d-serine, a physiological agonist of the NMDA receptor in mammalian brains. Schizosaccharomyces pombe produces serine racemase (spSR) that is highly similar to the brain enzyme. Our mass-spectrometric and X-ray studies revealed that spSR is modified with its natural sub ... >> More

  Serine racemase synthesizes d-serine, a physiological agonist of the NMDA receptor in mammalian brains. Schizosaccharomyces pombe produces serine racemase (spSR) that is highly similar to the brain enzyme. Our mass-spectrometric and X-ray studies revealed that spSR is modified with its natural substrate serine. spSR remains partially active even though its essential Lys57 inherently forming a Schiff base with the coenzyme pyridoxal 5'-phosphate is converted to N(6)-(R-2-amino-2-carboxyethyl)-l-lysyl (lysino-d-alanyl) residue. This indicates that the alpha-amino group of the d-alanyl moiety of the lysino-d-alanyl residue serves as a catalytic base in the same manner as the epsilon-amino group of Lys57 of the original spSR. << Less

  J. Biochem. 145:421-424(2009) [PubMed] [EuropePMC]

• Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe.

  Goto M., Yamauchi T., Kamiya N., Miyahara I., Yoshimura T., Mihara H., Kurihara T., Hirotsu K., Esaki N.

  D-serine is an endogenous coagonist for the N-methyl-D-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5'-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of L-serine to yield D-serine an ... >> More

  D-serine is an endogenous coagonist for the N-methyl-D-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5'-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of L-serine to yield D-serine and vice versa. The enzyme also catalyzes the dehydration of D- and L-serine. Both reactions are enhanced by Mg.ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 A resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with L-serine and D-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique "lysino-D-alanyl" residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-D-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme. << Less

  J. Biol. Chem. 284:25944-25952(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Inhibition of serine and proline racemases by substrate-product analogues.

  Harty M., Nagar M., Atkinson L., Legay C.M., Derksen D.J., Bearne S.L.

  d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formatio ... >> More

  d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (Ki=167±21mM, Ki'=661±81mM, cf. Km=19±2mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (Ki=111±15mM, cf. Km=5.7±0.5mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible. << Less

  Bioorg. Med. Chem. Lett. 24:390-393(2014) [PubMed] [EuropePMC]

• Catalytic mechanism of serine racemase from Dictyostelium discoideum.

  Ito T., Maekawa M., Hayashi S., Goto M., Hemmi H., Yoshimura T.

  The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were ... >> More

  The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of D- and L-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to L-serine dehydrase; S81A showed no racemase activity and had significantly reduced D-serine dehydrase activity, but it completely retained its L-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove D-serine dehydration by abstracting the α-hydrogen in D-serine. Our data suggest that the abstraction and addition of α-hydrogen to L- and D-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP. << Less

  Amino Acids 44:1073-1084(2013) [PubMed] [EuropePMC]