Systematic analysis for enzyme structures and functions in 2-hydroxyacid dehydrogenases

2-羟基酸脱氢酶的酶结构和功能的系统分析

• 批准号: 10660100
• 负责人: TAGUCHI Hayao
• 金额: $ 2.11万
• 依托单位: Science University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660100/
• 关键词: lactate dehydrogenase; stereochemistry; 2-hydroxyacid; allosteric enzyme; substrate specificity; alteration of enzyme function; catalytic center; three dimensional structure of protein; 乳酸菌; 立体構造解析; 鏡像異性体; 立体基質特異性

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.

戊糖乳酸脱氢酶（L.pentosus L-LDH）的2.3 nm分辨结构中发现了一种新的亚基间相互作用，这种作用可能与酶的非变构性质有关。X-射线衍射数据对干酪乳杆菌变构L-LDH的三维结构进行了精细化，最高可达2.4 nm。生化分析表明，这两个L-LDH始终表现出高的苹果酸脱氢酶活性，并在乳酸杆菌L-LDH中保守的Pro 101是部分负责这种广泛的底物特异性。粪肠球菌L-LDH的基因被克隆和测序，其表现出与干酪乳杆菌酶一样的共同的二价阳离子依赖性变构特性。该酶与干酪乳杆菌的酶具有特别高的序列同一性，序列比较表明这些L-LDH可能具有金属结合位点。戊糖乳杆菌D-LDH中Asn 97的氨基酸取代表明酶的主链原子参与了D-LDH的底物结合和催化作用。荧光分析表明，该酶结合辅酶和底物基本上是随机的，这表明与L-LDH的配体结合机制明显不同。从粪肠球菌（E.faecalis）细胞中分离纯化了两种不同分子量的D-扁桃酸脱氢酶，并对其进行了表征。