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

在参与碳氮键裂解的非蛋白水解酶中，有两种参与异腈代谢的酶如下所述。异腈水合酶是恶臭假单胞菌N19-2中催化异腈转化为n取代甲酰胺的一种新酶。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氨基酸序列与同源焦球菌的胞内蛋白酶（PH1704）相似度较低，且该蛋白酶的活性半胱氨酸残基在异腈水合酶的相应位置（Cys101）保守。一个含有Ala而不是Cys101的突变酶完全没有表现出异腈水合酶活性，证明了该残基在催化功能中的重要作用。在异腈代谢过程中，异腈水合酶使异腈水合生成n -取代甲酰胺。通过驯化培养从土壤中分离出一种微生物F164，进一步降解了前一种化合物。n -取代甲酰胺降解微生物鉴定为pascens节杆菌。发现微生物降解是通过酶促反应进行的，n取代的甲酰胺被水解生成相应的胺和甲酸酯。该酶被命名为n -取代甲酰胺去甲酰基酶（NfdA），并进行了纯化和表征。它通过化学计量催化n -苄基甲酰胺（NBFA: n -取代甲酰胺）水解为苄胺和甲酸酯。在所有测试的n取代甲酰胺中，NBFA是最合适的酶底物。然而，没有酰胺被接受作为底物。编码该酶的基因（nfdA）也被克隆。推导出的nfdA氨基酸序列在已知蛋白中与调控蛋白的整体序列一致性最高（28%）。只有NfdA的n端区域与酰胺水解酶超家族各成员的序列具有显著的同源性（27-73%），尽管除了上述有限区域外，整个序列没有相似性。少

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