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

植物激素、生长素生物合成相关酶和基因的研究

基本信息

批准号：
08456173
负责人：
KOBAYASHI Michihiko
金额：
$ 4.8万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456173/
关键词：
Plant hormone Indoleacetic acid Nitrilase Auxin Gene

项目摘要

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.

在蛋白质和基因水平上，分析了脂肪素粪中硝酸酶的硝酸酶，从吲哚-36-乙腈中形成植物性的脱糖酸。硝酸酶在大肠杆菌中过表达。为了研究该酶的羧基末端区域，该酶在迄今为止已知的各种硝酸酶中并非同源，我们在A.faecalis硝酸酶和犀牛Rhodococcus Rhodochrous J1之间构建了一组嵌合酶（或R.Rhodochrous K22）。每种嵌合酶也过表达，并显示出与野生a.faecalis硝酸酶相似的底物特异性，这表明该酶在底物特异性中的羧基区域不参与。没有羧基区域的突变酶显示出很少的硝酸酶活性，表明该区域将在蛋白质折叠或催化功能中起重要作用。另一方面，硝酸酶结构基因下游的1.5kb（发现使用E.Coli作为宿主的硝酸酶合成的异氟依依依依赖硝酸酶合成需要A.faecalis的nita）。该区域的序列分析揭示了984个核苷酸的开放阅读框（NITR）的存在。推导的NITR的氨基酸序列与阳性调节剂家族的氨基酸序列相似，包括来自putida putida的xyls和E.Coli的ARAC。发现NITR在NITA表达中为转录阳性调节剂编码。