Serine Racemase with Catalytically Active Lysinoalanyl Residue*

Yamauchi, Takae; Goto, Masaru; Wu, Hui Yuan; Uo, Takuma; Yoshimura, Teizo; Mihara, Hisaaki; Kurihara, Tatsuo; Miyahara, Ikuko; Hirotsu, Ken; Esaki, Nobuyoshi

doi:10.1093/jb/mvp010

Cited by 28 publications

(38 citation statements)

References 13 publications

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“…The only available experimentally determined structures of a eukaryotic serine racemase are four crystal structures of SR from fission yeast, S. pombe (PDB accession codes 2ZR8, 2ZPU, 1WTC, and 1V71) 14,16 . Like hSR, S. pombe SR acts as both a racemase and an eliminase and is activated by divalent cations and ATP.…”

Section: Multiple Alignment Analysismentioning

confidence: 99%

“…While the structure of a SR orthologue from the fission yeast Schizosaccharomyces pombe (35.1% 14 or 40% 15 sequence identity to human SR) has been solved 14,16 , the structure of a mammalian SR orthologue remains elusive. We therefore set out to establish a strategy for the generation and screening of human SR mutants in hopes of gaining insight into the structure-function relationships within the enzyme.…”

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confidence: 99%

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Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Hoffman

Jirásková

Zvelebil

et al. 2010

Collect. Czech. Chem. Commun.

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Human serine racemase (hSR) is a cytosolic pyridoxal-5′-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg 2+ cofactor as well as for overall stability of the enzyme. Keywords: D-Serine; Serine racemase; Error-prone PCR; Random mutagenesis; ThermoFluor assay; Enzymes; Racemases; Enzyme engineering; In vitro evolution.Eukaryotic serine racemase (SR; EC 5.1.1.18) is a cytosolic pyridoxal-5′-phosphate (PLP) dependent enzyme responsible for the production of D-serine from L-serine and vice versa. In the mammalian central nervous system (CNS), D-serine acts as an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors, which are involved in glutamate-mediated excitatory neurotrans-

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Section: Multiple Alignment Analysismentioning

confidence: 99%

mentioning

confidence: 99%

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Hoffman

Jirásková

Zvelebil

et al. 2010

Collect. Czech. Chem. Commun.

View full text Add to dashboard Cite

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“…Docking studies were carried out for the compounds 4-8 to verify whether they could be accommodated in the SR binding pocket. Crystal structures of SR have been recently solved [20][21][22] and the comparison between the holo SR structures and those in the presence of malonate revealed that the binding of the inhibitor triggers a transition from an open to a closed conformation. For this reason, in a previous work by us, SR conformational flexibility was investigated, identifying a few intermediate conformations that were included in our docking studies together with the open and closed conformations 33 .…”

Section: Resultsmentioning

confidence: 99%

“…In cells transfected with SR the pyruvate/D-serine ratio was found to be around 4, thus indicating that the a,b-elimination reaction might play a physiological role in the control of D-serine concentration 15 . Crystal structures of eukaryotic SR were solved from different sources, human, rat 20 and Schizosaccharomyces Pombe 21,22 , in holo form or in complex with the inhibitor malonate ( Figure 1). Despite the availability of structural data and the identification of a few inhibitors [23][24][25][26] , the development of specific and more potent SR inhibitors remains very challenging 13 .…”

Section: Introductionmentioning

confidence: 99%

Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target

Beato

Pecchini

Cocconcelli

et al. 2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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D-Serine is the co-agonist of NMDA receptors and binds to the so-called glycine site. D-Serine is synthesized by human serine racemase (SR). Over activation of NMDA receptors is involved in many neurodegenerative diseases and, therefore, the inhibition of SR might represent a novel strategy for the treatment of these pathologies. SR is a very difficult target, with only few compounds so far identified exhibiting weak inhibitory activity. This study was aimed at the identification of novel SR inhibitor by mimicking malonic acid, the best-known SR inhibitor, with a cyclopropane scaffold. We developed, synthesized, and tested a series of cyclopropane dicarboxylic acid derivatives, complementing the synthetic effort with molecular docking. We identified few compounds that bind SR in high micromolar range with a lack of significant correlation between experimental and predicted binding affinities. The thorough analysis of the results can be exploited for the development of more potent SR inhibitors.

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“…Therefore, among the ing that it is biosynthesized in C. elegans. In fact, Ser racemase (EC 5.1.1.16), a synthetic enzyme that produces D-Ser from L-Ser, has been found in various organisms, including yeast Schizosaccharomyces pombe, plants, and mammals (19,69,73). Moreover, in the C. elegans database WormBase (http://www.wormbase.org/), the gene T01H8.2 is annotated as encoding a putative Ser racemase.…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

Saitoh

Katane

Kawata

et al. 2012

Molecular and Cellular Biology

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(58,61). Its concentration in the blood of silkworms was reported to increase during particular stages of metamorphosis, although the physiological role of D-Ser in metamorphosis remains unclear (11). It is also present in the mammalian forebrain, where it persists at high concentrations throughout the life of the animal. DSer is now considered a neuromodulator that binds to the glycinebinding site of the N-methyl-D-Asp (NMDA) receptor, a subtype of the L-glutamate (L-Glu) receptor, and potentiates glutamatergic neurotransmission in the central nervous system (51,60,68). In fact, astroglia-derived D-Ser has been shown to regulate NMDA receptor-dependent long-term potentiation and/or long-term depression, which are basic processes of learning and memory, in the hypothalamic and hippocampal excitatory synapses (25,54). In addition, D-Ser is also found in the cerebellum during the early postnatal period, and it was recently reported that D-Ser derived from the Bergmann glia serves as an endogenous ligand for the ␦2 L-Glu receptor to regulate long-term depression at synapses between parallel fibers and Purkinje cells in the immature cerebellum but not the mature one (31). These lines of evidence suggest the physiological significance of D-Ser in the regulation of higher brain functions through the L-Glu receptors, and indeed, perturbation of D-Ser levels in the central nervous system has now been implicated in the pathophysiology of various neuropsychiatric disorders, including schizophrenia (4, 23, 24, 71), Alzheimer's disease (28, 70), and amyotrophic lateral sclerosis (59).Unlike the tissue-specific expression of D-Ser, substantial amounts of free D-Asp are present in a wide variety of tissues and cells in invertebrates and vertebrates, particularly in the central nervous, neuroendocrine, and endocrine systems. D-Asp is proposed to play an important role in regulating developmental processes, hormone secretion, and steroidogenesis (12,26,35). For instance, it has been reported that in male lizards, intraperitoneally administered D-Asp is taken up rapidly by the testis, which induces a significant increase in testosterone levels and a significant decrease in 17␤-estradiol levels in the testis and plasma (57). Interestingly, a reverse relationship is found between males and females; specifically, in female lizards, exogenous D-Asp induces a significant decrease in testosterone levels in the ovary and plasma, while it enhances follicular production of 17␤-estradiol by upregulating the local aromatase activity (2). Similar findings have also been reported in studies of the green frog (16, 56). These observations suggest that D-Asp is an important regulatory molecule of gonads. In addition, it was recently shown that the amount of D-Asp in human seminal plasma and spermatozoa is significantly reduced in oligoasthenoteratospermic and/or azoospermic donors compared with normospermic donors (14). Moreover, in human female patients undergoing in vitro fertilization, the D-Asp content of the preovulatory follicular fluid ...

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Serine Racemase with Catalytically Active Lysinoalanyl Residue*

Cited by 28 publications

References 13 publications

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target

Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

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