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

从蛋白和基因水平分析了粪Alcaligenes faecalis中由吲哚-36-乙腈生成植物激素吲哚乙酸的腈酶。硝化酶在大肠杆菌中过表达。为了研究该酶的羧基末端区域，我们构建了一组粪A.faecalis硝化酶与Rhodococcus rhodochrous J1（或r.r odochrous K22）之间的嵌合体酶。每一种嵌合体酶也都过表达，并表现出与野生粪A.faecalis nitrilase相似的底物特异性，表明该酶的羧基区不参与底物特异性。没有羧基区域的突变酶表现出极低的腈酶活性，表明该区域在蛋白质折叠或催化功能中起重要作用。另一方面，我们发现粪芽孢杆菌的硝化酶结构基因（nitA）下游1.5kb是大肠杆菌作为宿主诱导异戊腈依赖的硝化酶合成所必需的。序列分析显示，该区域存在一个984个核苷酸组成的开放阅读框（nitR）。推断出的nitR的氨基酸序列与恶臭假单胞菌的XylS和大肠杆菌的AraC等阳性调节家族相似。发现nitR编码nitA表达的转录阳性调节因子。

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.