Novel isonitrile hydratase involved in isonitrile metabolism.
We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726-13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent K(m) value for cyclohexyl isocyanide was 0.95 ± 0.05 mm. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH(4))(2)SO(4) as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB.
Sato
Hiroyoshi
H
Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Hashimoto
Yoshiteru
Y
Fukatsu
Hiroshi
H
Kobayashi
Michihiko
M
D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D003486:Cyanides / Q000737:chemistry* / Q000378:metabolism; D004790:Enzyme Induction; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D003486:Cyanides; D006836:Hydro-Lyases
J Biol Chem. 1992 Oct 15;267(29):20746-51
1400390
Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):714-8
11607511
Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13726-31
15358859
Nat Prod Rep. 1988 Jun;5(3):229-45
3067133
J Biol Chem. 1996 Jun 28;271(26):15796-802
8662959
Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):14849-54
18809911
J Biol Chem. 2005 Mar 11;280(10):8660-7
15632196
J Bacteriol. 1990 Sep;172(9):4807-15
2394676
Nature. 1970 Aug 15;227(5259):680-5
5432063
J Biol Chem. 2002 Nov 29;277(48):45860-5
12244065
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4267-72
8633053
Anal Biochem. 1976 May 7;72:248-54
942051
Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):564-8
16407114
J Biol Chem. 2003 Aug 8;278(32):29600-8
12773527
Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):247-51
8419930
Trends Biotechnol. 1992 Nov;10(11):402-8
1368882
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10572-7
8855219
J Biol Chem. 2001 Jun 29;276(26):23480-5
11306561
J Biol Chem. 2009 May 29;284(22):14930-8
19346246
Nat Biotechnol. 1998 Aug;16(8):733-6
9702770
Biosci Biotechnol Biochem. 2005 Jan;69(1):228-30
15665493