|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1999 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1998 : ¥2,400,000 (Direct Cost : ¥2,400,000)
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.