Function and Structure of NADP-Dependent D-Amino Acid Dehydrogenase

一株能产生依赖NADP的D-苯丝氨酸脱氢酶的土壤细菌被鉴定为丁香假单胞菌。该酶催化D-苏氨酸-3-苯基丝氨酸的3-羟基的氧化。从丁香菇NK-15的粗提物中纯化得到了均一的酶，并与十字青霉的依赖NADP的D-苏氨酸脱氢酶的性质进行了比较。其相对分子质量、亚基组成和最适pH等性质与D-苏氨酸脱氢酶相似。然而，这两种酶的热稳定性、底物专一性、辅因子专一性和抑制剂是不同的。N端27个氨基酸序列表明NADP结合位点位于酶的N端区域。为了确定这两种酶的一级结构，用菌落杂交的方法克隆了这两种酶的基因。未获得同时具有这两种酶基因的克隆。根据所分离的多肽的氨基酸序列以及N-端和C-端的氨基酸序列合成引物，进行聚合酶链式反应。用pUC18将扩增的D-苏氨酸脱氢酶基因片段克隆到大肠杆菌JM109中。将D-苯丝氨酸脱氢酶基因的扩增产物克隆到E.ColiJM109中，对插入的DNA进行测序。虽然无法确定两种酶的完整一级结构，但两种酶的N-端和C-端氨基酸序列并不相似。

A soil bacterium which produced an inducible NADP-dependent D-phenylserine dehydrogenase was identified as Pseudomonas syringae. The enzyme catalyzed the oxidation of the 3-hydroxyl group of D-threo-3-phenylserine. The enzyme was purified to homogeneity from a crude extract of P.syringae NK-15 and its properties were compared with those of a constitutive NADP-dependent D-threonine dehydrogenase from P.cruciviae. The properties such as molecular weight, subunit composition, and optimum pH were similar to those of D-threonine dehydrogenase. However, thermal stability, substrate specificity, cofactor specificity, and inhibitors were defferent in both enzymes. The N-terminal 27 amino acids sequence suggested that the NADP-binding site was located in the N-terminal region of the enzyme. To determine primary structure of both enzymes, cloning of both enzyme genes was carried out using colony hybridization. Clones having both enzyme geneswere not obtained. PCR was carried out using primers synthesized according to the amino acid sequence of the isolated peptide and N- and C-terminal amino acid sequences. The amplified DNA fragments of D-threonine dehydrogenase gene was cloned into E.coli JM109 using pUC18. The PCR product of D-phenylserine dehydrogenase gene was cloned into E.coli JM109 and the sequence of the inserted DNA was determined. Although the complete primary structure of both enzymes could not be determined, N-terminal and C-terminal amino acid sequences were not similar in both enzymes.