| Project/Area Number |
06454077
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SODA Kenji Institute for Chemical Research, Kyoto University Professor, 化学研究所, 教授 (30027023)
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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)
ESAKI Nobuyoshi Institute for Chemical Research, Kyoto University Associate Professor, 化学研究所, 助教授 (50135597)
吉村 徹 京都大学, 化学研究所, 助手 (70182821)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1995: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1994: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | D-Amino acid / Alanine Racemase / D-Amino acid aminotransferase / Glutamate racemase / Suicide substrate / 酵素自殺基質 / UDP-N-アセチルムラミル-L-アラニン / ピリドキサルリン酸 / D-アミノ酸トランスアミナーゼ / セリンO-スルフェート / 酵素阻害剤 |
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| Research Abstract |
D-Amino acids such as D-alanine and D-glutamate are the indespensable components of the peptidoglycan layr of the bacterial cell walls. Thus, the specific inhibitors for the bacteral enzymes participate in the biosyntheses of D-amino acids can be potent antibiotics. We studied detailed mechanisms of alanine racemase, D-amino acid aminotransferase, and glutamate racemase, to develop the meachanism-based inhibitors for these enzymes. We studied the role of Lys39 of the thermostable alanine racemase of Bacillus stearothermophilus, which is bound to the cofactor, pyridoxal 5'-phosphate (PLP) , and suggested this residue to be a catalytic base by site-directed mutagenesis. The terminal amino group of Lys39 acts as a base to abstract the 2-hydrogen from the substrate. The effects of alkylamines on the K39A mutant enzymes suggest that Lys39 acts as a sole catalytic base to abstract the 2-hydrogen from the substrate and returns it to the 2-carbon of substrate moiety of a deprotonated intermedi
… More
ate. D-Amino acid aminotransferase catalyzes the transfer of amino group between various D-amino acids and keto acids. We studied the catalytic role of leucine 201 residue of the themostable D-amino acid aminotransferase : the residue was crystallographically shown to be in the vicinity of the active-site to interact with the bound PLP by site-directed mutagenesis. The Leu 201 residue probably regulates the function of cofactor during the conversion of PMP to PLP.Glutamate racemase catalyzes the racemization of glutamate to produce D-glutamate. We compared the enzyme with those of Lactic acid bacteria. The addition of UDP-N-acetylmuramyl-L-alanine, an activator of the E.coli enzyme affected the CD and fluorescence spectra of the E.coli enzyme. In contrast, the enzymes of P.pentosaceus and L.brevis were not activated by UDP-N-acetylmuramyl-L-alanine. The Pediococcus enzyme shows a significant sequence similarity to mammalian myoglobin, and was inhibited by hemin. Glutamate racemase of E.coli was not inhibited by hemin in the absence of UDP-N-acetylmuramyl-L-alanine, but strongly inhibited in the presence of UDP-N-acetylmuramyl-L-alanine. The conformation of the E.coli enzyme is converted to a similar form to that of the Pediococcus enzyme by the addition of UDP-N-acetylmuramyl-L-alanine. Less
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