1999 Fiscal Year Final Research Report Summary
Systematic analysis for enzyme structures and functions in 2-hydroxyacid dehydrogenases
Project/Area Number |
10660100
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
応用微生物学・応用生物化学
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Research Institution | Science University of Tokyo |
Principal Investigator |
TAGUCHI Hayao Faculty of Science and Technology, Science University of Tokyo, 理工学部, 助教授 (90188136)
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Project Period (FY) |
1998 – 1999
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Keywords | lactate dehydrogenase / stereochemistry / 2-hydroxyacid / allosteric enzyme / substrate specificity / alteration of enzyme function / catalytic center / three dimensional structure of protein |
Research Abstract |
In L.pentosus L-LDH, novel type of intersubunit interactions for L-LDHs, which may be involved in the non-allosteric properties of the enzyme, were found in the 2.3 Å resolution structure of the enzyme. The 3-D structure of L.casei allosteric L-LDH is being refined the X-ray diffraction data up to 2.4 Å. Biochemical analysis showed that both the two L-LDHs consistently exhibit high malate dehydrogenase activity, and that conserved Pro101 in Lactobacillus L-LDHs is partially responsible for such a broad substrate specificity. The gene for E.faecalis L-LDH, which exhibit a common divalent cation-dependent allosteric properties like the L.casei enzyme, was cloned and sequenced. The enzyme showed a particularly high sequence identity with the L.casei enzyme, and the sequence comparison suggested possible metal-binding sites of these L-LDHs. For L.pentosus D-LDH, amino acid substitution of Asn97 indicated that main chain atoms of the enzyme are involved in the substrate binding and catalysis of D-LDH. Fluorescence analysis revealed that the enzyme binds coenzyme and substrate essentially randomly, indicating markedly different ligand binding mechanisms from those of L-LDHs. D-Mandelate dehydrogenases were purified from E.faecalis cell, which was shown to possess two types of the enzymes with distinct molecular weights, and then characterized.
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