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

从蛋白质和基因水平分析了从吲哚-36-乙腈生成植物荷尔蒙吲哚乙酸的粪产碱菌中的腈酶。在大肠埃希菌中高效表达了该酶。为了研究该酶的羧基末端区域，我们构建了一组粪便酵母酶与红球菌J1(或红球菌K22)之间的嵌合酶。每一种Chimira酶也都过表达，并表现出与野生粪腈酶相似的底物专一性，这表明该酶的羧基区域不参与底物专一性。该突变酶不具有羧基区，显示出很低的酶活性，表明该区在蛋白质折叠或催化功能中起重要作用。另一方面，粪链霉腈酶结构基因(NITA)下游1.5kb的片段被发现是依赖异戊腈诱导以大肠杆菌为宿主的氰化酶合成所必需的。对该区域的序列分析表明，存在一个由984个核苷酸组成的开放阅读框架(NitR)。NitR的氨基酸序列与恶臭假单胞菌的XylS和大肠杆菌的AraC等正性调控家族的氨基酸序列相似。在NITA的表达中，NitR被发现编码转录正调控因子。

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.