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Functional analysis of non-proteolytic enzymes involved in carbon-nitrogen bond cleavage

参与碳氮键断裂的非蛋白水解酶的功能分析

基本信息

批准号：
14360047
负责人：
KOBAYASHI Michihiko
金额：
$ 9.41万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Among non-proteolytic enzymes involved in carbon-nitrogen bond cleavage, two enzymes involved in isonitrile metabolism are described as follows.Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, the isonitrile hydratase gene was cloned. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys101). A mutant enzyme containing Ala instead of Cys101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.N-Substituted formamide was produced through the hydration of an isonitrile by isonitrile hydratase in the isonitrile metabolism. The former compound was further degraded by a … More microorganism, F164, which was isolated from soil through an acclimatization culture. The N-substituted formamide-degrading microorganism was identified as Arthrobacter pascens. The microbial degradation was found to proceed through an enzymatic reaction, the N-substituted formamide being hydrolyzed to yield the corresponding amine and formate. The enzyme, designated as N-substituted formamide deformylase (NfdA), was purified and characterized. It stoichiometrically catalyzed the hydrolysis of N-benzylformamide (NBFA : an N-substituted formamide) to benzylamine and formate. Of all the N-substituted formamides tested, NBFA was the most suitable substrate for the enzyme. However, no amides were accepted as substrates. The gene (nfdA) encoding this enzyme was also cloned. The deduced amino acid sequence of nfdA exhibited the highest overall sequence identity (28%) with those of regulatory proteins among known proteins. Only the N-terminal region of NfdA also showed significant sequence identity (27-73%) to that of each member of the amidohydrolase superfamily, although there was no similarity in the overall sequence except in the above limited region. Less

在参与碳-氮键断裂的非蛋白水解酶中，有两种酶参与异腈代谢，异腈水合酶是恶臭假单胞菌N19-2中的一种新酶，催化异腈转化为N-取代甲酰胺。根据N端和内部氨基酸序列，克隆了异腈水合酶基因。预测的inhA的氨基酸序列与Pyrococcus horikoshii（PH 1704）的胞内蛋白酶的氨基酸序列相似性较低，并且该蛋白酶中的活性半胱氨酸残基在异腈水合酶中的相应位置（Cys 101）是保守的。含有Ala而不是Cys 101的突变体酶没有表现出异腈水合酶活性在所有，证明了这个残基的催化function. N-取代甲酰胺的催化function. N-取代甲酰胺通过异腈水合酶在异腈代谢中的异腈的水合作用。前一种化合物进一步降解， ...更多信息通过驯化培养从土壤中分离得到一株微生物F164。N-取代甲酰胺降解菌经鉴定为节杆菌（Arthrobacter pascens）。发现微生物降解通过酶促反应进行，N-取代甲酰胺被水解以产生相应的胺和甲酸盐。该酶，命名为N-取代甲酰胺脱甲酰基酶（NfdA），纯化和表征。它化学计量催化N-苄基甲酰胺（NBFA：一种N-取代甲酰胺）水解为苄胺和甲酸酯。在所有的N-取代的甲酰胺测试，NBFA是最合适的酶的底物。然而，没有酰胺被接受为底物。还克隆了编码该酶的基因（nfdA）。推导的nfdA的氨基酸序列与已知蛋白中的调节蛋白具有最高的整体序列同一性（28%）。只有NfdA的N-末端区域也显示出与酰胺水解酶超家族的每个成员的显著序列同一性（27-73%），尽管除了上述有限区域之外，在整个序列中没有相似性。少