JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M208571200 on September 18, 2002

J. Biol. Chem., Vol. 277, Issue 48, 45860-45865, November 29, 2002
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Isonitrile Hydratase from Pseudomonas putida N19-2
CLONING, SEQUENCING, GENE EXPRESSION, AND IDENTIFICATION OF ITS ACTIVE AMINO ACID RESIDUE*

Masahiko GodaDagger §**, Yoshiteru HashimotoDagger **, Masanori TakaseDagger , Sachio HeraiDagger , Yasuhito IwaharaDagger , Hiroki HigashibataDagger , and Michihiko KobayashiDagger ||

From the Dagger  Institute of Applied Biochemistry, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and § Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

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.

* This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by an Industrial Technology Research Grant Program in 2002 from New Energy and Industrial Technology Development Organization (NEDO) of Japan, by The Agricultural Chemical Research Foundation, by National Project on Protein Structural and Functional Analyses, and by Research Grant (A) of the University Research Projects.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Both authors contributed equally to the results of this work.

Research fellow of the Japan Society for the Promotion of Science.

|| To whom correspondence should be addressed. Tel.: 81-298-53-4628; Fax: 81-298-53-4605.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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