Studies on enzymes and genes involved in biosynthesis of plant hormone, auxin

从蛋白质和基因水平对粪产碱菌腈水解酶进行了分析。腈水解酶在大肠杆菌中得到高效表达。为了研究该酶的羧基末端区域，这是迄今已知的各种腈水解酶中不同源的，我们构建了一组粪曲霉腈水解酶和玫瑰色红球菌J1（或玫瑰色红球菌K22）之间的嵌合酶。每种chimira酶也过表达，并显示出与野生粪肠球菌腈水解酶相似的底物特异性，表明酶的羧基区域不参与底物特异性。缺失羧基的突变体酶活性很低，表明该区域在蛋白质折叠或催化功能中起重要作用，而粪曲霉腈水解酶结构基因（nitA）下游1.5kb的区域是大肠杆菌腈水解酶合成所必需的。该区域的序列分析显示存在由984个核苷酸组成的开放阅读框架（nitR）。推导的nitR的氨基酸序列与恶臭假单胞菌的XylS和大肠杆菌的AraC等正调控因子家族的氨基酸序列相似。发现nitR编码nitA表达的转录正调控因子。

Nitrilase in Alcaligenes faecalis, which forms the plant horomone indoleacetic acid from indole-36-acetonitrile, were analyzed at both protein and gene levels. The nitrilase was overexpressed in Escherichia coli. In order to investigate the carboxyl terminal region of this enzyme which is not homologous among various nitrilases so far known, we constructed a set of chimera enzymes between the A.faecalis nitrilase and Rhodococcus rhodochrous J1 (or R.rhodochrous K22). Each chimira enzyme was also overexpressed and showed similar substrate specificity to that of the wild A.faecalis nitrilase, suggesting the non-involvement of the carboxyl region of the enzyme in substrate specificity. The mutant enzyme that does not have the carboxyl region exhibited a very little nitrilase activity, indicating that this region would play an important role in the protein folding or the catalytic function.On the other hand, the 1.5kb downstream from the nitrilase structural gene (nitA) of A.faecalis was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis using E.coli as a host. Sequence analysis of this region revealed the existence of an open reading frame (nitR) composing of 984 nucleotides. The deduced amino acid sequence of nitR showed similarity to that of a positive regulator family including XylS from Pseudomonas putida and AraC from E.coli. The nitR was found to code for a transcriptional positive regulator in nitA expression.