Studies on enzymes and genes involved in biosynthesis of plant hormone, auxin
Studies on enzymes and genes involved in biosynthesis of plant hormone, auxin
批准号:
08456173
负责人:
KOBAYASHI Michihiko
金额:
$4.8万
依托单位:
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
项目状态:
已结题
起止时间:
1996 至 1997
中文摘要
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。
英文摘要
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.
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小林 達彦: "重金属コバルトによるスーパー酵素の誘導" バイオサイエンスとインダストリー. 55. 208-209 (1997)
小林达彦:“重金属钴诱导超级酶”《生物科学与工业》55. 208-209 (1997)。
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M.Kobayashi: "Development of catalytic functions of nitrile-converting enzymes for production of useful compounds." Nippon Nogeikagaku Kaishi. 71. 1243-1252 (1997)
M.Kobayashi:“开发用于生产有用化合物的腈转化酶的催化功能。”
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Komeda,H.,Kobayashi,M.&Shimizu,S.: "A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L‐NHase) induced by its reaction product" J.Biol.Chem.271. 15796‐15802 (1996)
Komeda, H.、Kobayashi, M. 和 Shimizu, S.:“一种新的基因簇,包括红球菌 J1 nhlBA 基因,编码由其反应产物诱导的低分子量腈水合酶 (L-NHase)” J.Biol.Chem .271.15796-15802(1996)
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共 25 条
Studies on unique inducible protein expression system for streptomycetes
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Microbial degradation of bioactive substance from turmeric
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Interdisciplinary analysis of Japan's experience of the First World War: using new primary resources
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Characterization of an N3 compound-degrading enzyme
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Comprehensive elucidation and application of the function of enzymes involved in cleavage and synthesis of a carbon-nitrogen bond
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Analyses of the N-substituted formamide metabolic pathway and their applications to the production of useful compounds
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Functional analysis of non-proteolytic enzymes involved in carbon-nitrogen bond cleavage
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Development of Rhodococcus promoter for utilization in actinomycetes
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资助金额:$8.9万
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财政年份:2001
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Development of novel expression vectors utilizing gene promoters involved in nitrile metabolism
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批准号:07556073
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项目类别:Grant-in-Aid for Scientific Research (A)
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资助金额:$4.1万
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财政年份:1995
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负责人:KOBAYASHI Michihiko
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依托单位:
Molecular functional analysis of gene cluster involved in nitrile metabolism
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批准号:06660101
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$1.34万
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财政年份:1994
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负责人:KOBAYASHI Michihiko
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依托单位:
海外基金