title: Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue.
abstract: 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.
authors.affiliation: Institute of Applied Biochemistry, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Goda
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Masanori
authors.initials: M
authors.lastname: Takase
authors.forename: Sachio
authors.initials: S
authors.lastname: Herai
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Iwahara
authors.forename: Hiroki
authors.initials: H
authors.lastname: Higashibata
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C048831:nitrile hydratase