Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue.
Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue.
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来自恶臭假单胞菌 N19-2 的异腈水合酶。
DOI：
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发表时间：
2002
影响因子：
4.8
通讯作者：
Michihiko Kobayashi
中科院分区：
文献类型：
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作者：
M. Goda;Y. Hashimoto;M. Takase;Sachio Herai;Yasuhito Iwahara;H. Higashibata;Michihiko Kobayashi
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，扩增出与异腈水合酶基因部分对应的535 bp DNA片段，作为探针，克隆出包含整个基因的6.4 kb DNA片段。序列分析显示，异腈水合酶基因（inhA）全长684个核苷酸，编码一个分子量为24,211 Da的蛋白。在大肠杆菌中过量表达inhA可产生大量可溶性异腈水合酶，其分子和催化性能与假单胞菌的天然酶相同。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，且在异腈水合酶的相应位置（Cys-101）上保留了一个活性半胱氨酸残基。一个含有Ala而不是Cys-101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。
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, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. 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 (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.
影响因子：
3.5
作者：
HO, SN;HUNT, HD;PEASE, LR
通讯作者：
PEASE, LR