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

一种产生nadp依赖性d -苯基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 -三苯基丝氨酸的3-羟基氧化。从丁香p . NK-15粗提物中纯化出该酶，并将其性质与十字花科p . nadp依赖性d -苏氨酸脱氢酶进行了比较。其分子量、亚基组成、最适pH值等性质与d -苏氨酸脱氢酶相似。然而，两种酶的热稳定性、底物特异性、辅因子特异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初级结构，利用集落杂交技术克隆了这两种酶的基因。没有获得具有这两种酶基因的克隆。根据分离肽的氨基酸序列和N端、c端氨基酸序列合成引物进行PCR。将扩增得到的d -苏氨酸脱氢酶基因片段用pUC18克隆到大肠杆菌JM109中。将d -苯基丝氨酸脱氢酶基因的PCR产物克隆到大肠杆菌JM109中，并测定了插入DNA的序列。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.