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

在L乳酸脱氢酶中，在2.3Å分辨结构中发现了L-乳酸脱氢酶的新型亚基间相互作用，这可能与该酶的非变构性质有关。利用X射线衍射仪对干酪乳杆菌L-乳酸脱氢酶的三维结构进行了细化，得到了高达2.4Å的X射线衍射数据。生化分析表明，两株L-LDHs均表现出较高的苹果酸脱氢酶活性，而L-LDHs中保守的Pro101是产生如此广泛的底物专一性的部分原因。粪肠球菌L-乳酸脱氢酶具有与酪乳杆菌酶相似的常见的二价阳离子依赖变构性质，该基因被克隆和测序。该酶与酪乳杆菌的同源性特别高，序列比较表明这些L-乳酸脱氢酶可能具有金属结合部位。对于D-乳酸脱氢酶，Asn97的氨基酸取代表明该酶的主链原子参与了D-乳酸脱氢酶的底物结合和催化。荧光分析表明，该酶与辅酶和底物的结合基本上是随机的，这表明该酶与L-乳酸脱氢酶的配体结合机制明显不同。从粪肠球菌细胞中分离纯化了D-扁桃酸脱氢酶，发现其具有两种不同分子量的酶，并对其进行了鉴定。

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.