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

Fujitani, Yoshiyuki; Horiuchi, T; Ito, Kazutoshi; Sugimoto, Mineharu

doi:10.1016/j.phytochem.2007.03.040

Cited by 31 publications

(35 citation statements)

References 26 publications

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“…(Cook et al 2002;De Miranda et al 2002). Such activation has also been observed in plant enzymes (Fujitani et al 2006;Fujitani et al 2007;Yoshimura and Goto 2008). Alanine racemase is a highly specific amino acid racemase (Kino et al2007).…”

Section: Effect Of Temperature and Ph On Enzyme Activitymentioning

confidence: 83%

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Liu

Shi

2011

World J Microbiol Biotechnol

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Alanine racemase catalyzes the interconversion of D: - and L: -alanine and plays an important role in supplying D: -alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET-alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His(6)-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni(2+)-NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with L: -alanine and L: -isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0-11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr(2+), Mn(2+), Co(2+), and Ni(2+) obviously enhanced enzymatic activity, while the Cu(2+) ion showed inhibitory effects.

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Section: Effect Of Temperature and Ph On Enzyme Activitymentioning

confidence: 83%

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Liu

Shi

2011

World J Microbiol Biotechnol

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“…All eight homologues included in our alignment catalyze both serine racemization and elimination and are activated by Mg 2+ ions. Most of these racemases are also activated by ATP, with the exception of SR orthologues from Hordeum vulgare (barley) 33 and Arabidopsis thaliana 34 . Mammalian SRs exhibit a very high degree of sequence identity (84-91%), and we recently published a comparative study of the human and mouse orthologues 17 .…”

Section: Multiple Alignment Analysismentioning

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.

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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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“…Recently, investigation of different Arabidopsis accessions has revealed considerable natural variation in uptake and metabolism of D-amino acids (29,30). Identification of plant genes encoding D-amino acid metabolism enzymes such as D-amino acid oxidases (31), D-amino acid transaminases (32), and particularly the D-Ser and D-Ala racemases (33)(34)(35)(36) provides clear evidence for endogenous D-amino acid biosynthesis and metabolism, implying that D-amino acids may have important biological functions in plants. This suggestion was substantiated by the finding that D-Ser activates glutamate receptors and plays a role in pollen tube growth and morphogenesis (37).…”

Section: Discussionmentioning

confidence: 99%

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

Strauch

Svedin

Dilkes

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. As an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolitegene associations led to the identification of N-malonyl-D-alloisoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5′-phosphate for its activity. This study thus identifies a new D-amino acid racemase gene family and advances our knowledge of plant Damino acid metabolism that is currently largely unexplored. It also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.D-amino acid | racemase | genome-wide association | secondary metabolism | natural variation P lants have the ability to create over 200,000 small compounds known as secondary or specialized metabolites (1). These chemically diverse compounds help mediate plant adaptation to their environment and play important roles in plant defense mechanisms, pigmentation, and development. In addition, many of these metabolites are desirable to humans as medicinal and nutritional compounds. Therefore, furthering our understanding of plant specialized metabolism will have profound impacts on various applications from crop improvement to human health.To date, only a small fraction of the chemical and catalytic space in plant specialized metabolism has been explored. Even in the best-studied model plant Arabidopsis thaliana, there are still many uncharacterized metabolites, and the vast majority of genes encoding enzymes implied to be involved in specialized metabolism do not have known associations with any metabolites. Several studies of Arabidopsis natural accessions (individuals collected from wild populations) revealed considerable qualitative and quantitative variation in the accumulation of various compounds such as glucosinolates, terpenoids, and phenylpropanoids (2-4). This extensive metabolite variation can be attributed to genetic variation in genes encoding enzymes and regulatory factors of the pathways involved; quantitative trait locus (QTL) mapping has successfully uncove...

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

Cited by 31 publications

References 26 publications

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

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

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

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