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

在碳氮键裂解的非蛋白质酶中，腹膜上水合物是一种新的酶，在pseudomonas putida n19-2中，它催化了异构体的转化为n-盐酸的正式和内部氨基酸序列。 . o that of an intracellular Protease in Pyrococcus Horikoshii (PH1704), and An Active Cysteine Residue in The Protease Was Consusvered in THE ISONITRILE HYDRATASE AT TORRESPONDING POSITION (CYS101). Ivity at all, the essential role of this. Of An IsonitRile在异构体的代谢中，以前的综合酶是由溶解的，从土壤中溶解的。将N-取代的甲酰胺水解为相应的酶，被指定为N-取代的甲酰胺变形酶（NFDA），并表征了N-Benzylfa（NBFA）：NBFA：NBFA。但是，作为底物的甲酸盐。（27-73％）降低了酰胺水解酶超家族的每个成员，尽管整体序列中没有相似性，但区域较少