Alternation in Substrate Specificity of Amino Acid Dehydrogenases based on the Structural Analyses

通过对共生梭菌谷氨酸脱氢酶（GluDH）的结构分析，研究了球形芽孢杆菌苯丙氨酸脱氢酶（PheDH）对亮氨酸脱氢酶（LeuDH）底物特异性的变化。我们关注的是底物口袋周围的氨基酸脱氢酶中不常见的残基。GluDH和LeuDH中的163A和377V在PheDH中分别变为G和L。修饰后的GluDH对l -苯丙氨酸的活性降低，对脂肪族氨基酸的活性提高。从土壤分离的节肢杆菌菌株1C中发现的Opine脱氢酶（ODH）与植物冠瘿中发现的40 kda蛋白、d -溶血碱脱氢酶、d -野碱脱氢酶具有相似性。从大肠杆菌转化体中高度纯化出ODH。在辅助因子和底物存在或不存在的情况下获得的ODH晶体已被证明可以衍射到1.8Å以上的分辨率。我们对这些残基和这两个结构域的其他残基进行了定点突变，结果表明，这6个残基中的任何一个或Arg-143、Lys-156、His-222或Tyr-280的取代都显著损害了催化作用。稳态动力学相互作用和催化。一种类似物N-[1-(S)-（羧基）乙基]-(S)-苯丙氨酸竞争性地抑制了野生型和除Arg143突变体外的大多数突变体。此外，我们在His-202、Tyr-222、Arg-292和Tyr-293的推测活性位点附近观察到硫酸盐离子结合。我们模拟了硫酸结合位点的丙酮酸，并提出了ODH类似于2-羟基酸脱氢酶的催化机制。

According to the structural analysis of glutamate dehydrogenase (GluDH) of Clostridium symbiosum, we studied the alternation of substrate specificity of phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus to leucine dehydrogenase (LeuDH). We focused on residues which are not common in the amino acid dehydrogenases around the pocket for the substrate. 163A and 377V which are found in GluDH and LeuDH were changed to G and L in PheDH, respectively. The modified GluDH showed lower activity toward L-Phe, where as the activity toward aliphatic amino acids were increased.Opine dehydrogenase (ODH) from a soil isolate Arthrobacter sp. strain 1C showed similarity toward 40-kDa Protein, D-lysopine dehydrogenase, D-nopaline dehydrogenase found in plant crown gall. ODH was highly purified from E. coli transformant. Crystals of ODH, obtained in the presence or absence of co-factor and substrate, have been shown to diffract to beyond 1.8Å resolution. We carried out site-directed mutagenesis on these residues and other ones in both domains, and the results indicated that substitution of either of these six residues or Arg-143, Lys-156, His-222, or Tyr-280 impaired catalysis significantly. Steady-state kinetic interaction and catalysis. An opine analog N-[1-(S)-(carboxyl)ethyl]-(S)-phenylalanine competitively inhibited the wild type and most mutants with the exception of Arg143 mutants, Further, we observed a sulfate ion bound in the vicinity of His-202, Tyr-222, Arg-292 and Tyr-293 in the putative active site. We modeled a pyruvate in the sulfate binding site and a catalytic mechanism of ODH to resemble that of the 2-hydroxy acid dehydrogenases is proposed.