1995 Fiscal Year Final Research Report Summary
Biochemical and Molecular Biological Studies or Structure and Function of Glutamate Racemase
| Project/Area Number |
06680610
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional biochemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ESAKI Nobuyoshi Institute for Chemical Research, Kyoto University Associate Professor, 化学研究所, 助教授 (50135597)
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| Co-Investigator(Kenkyū-buntansha) |
KURIHARA Tatsuo Institute for Chemical Research, Kyoto University Instructor, 化学研究所, 助手 (70243087)
YOSHIMURA Tohru Institute for Chemical Research, Kyoto University Instructor, 化学研究所, 助手 (70182821)
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| Project Period (FY) |
1994 – 1995
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| Keywords | glutamate racemase / gene cloning / Bacillus cereus / cysteine residue / composite active site / alpha, beta-elimination / suicide substrate / serine O-sulfate |
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| Research Abstract |
The glutamate racemase gene of P.pentosaceus consists of a 795-nucleotides open reading frame, and encodes 265 amino acid residues which form a monomeric protein. The sequence shows significant homology with that of aspartate racemase from S.thermophilus : it requires no cofactors and contains an essential cysteinyl residue. The two racemases are structurally similar to each other, in particular in the regions around the two cysteinyl residues. We have also found significant sequence homology between glutamate racemase and mammalian myoglobins, in particular in the regions corresponding to a heme binding procket of myoglobins. Glutamate racemase is bound with an equimolar amount of hemin to be inactivated, and aspartate racemase shows a low sequence homology with myoglobins, but is not bound with hemin. These suggest that glutamate racemase may be derived from an evolutionary origin of globin family proteins. Aspartate racemase also may have evolved from the common ancestral protein, b
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ut its structure may have been altered more extensively than glutamate racemase by divergence. We have shown that the amino acid sequence deduced from the nucleotide sequence of murI (dga) gene that is required for the biosynthesis of D-glutamate as an essential component of peptidoglycan in E.coli has significant homology with that of glutamate racemase of P.pentosaceus. The gene was ligated into a plasmid, pKK223-3 with a designed ribosome binding site, and expressed in E.coli JM109 cells. Glutamate racemase was produced in the transformant cells, whereas the enzyme was not found in the host cells. I partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000. We have also found three highly conserved regions in the glutamate racemase genes previously sequenced and shown that their conserved regions exsist on the chromosomes of various kinds of bacterial strains. These indicate that D-glutamate, which is indispensable for almost all bacterial strains as a constituent of peptidoglycans, is directly produced from its L-enantiomer by glutamate racemase. Less
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