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.We have extensively studied the biological metabolism of toxic compounds containing a CϵN moiety, such as nitriles (1-5) and isonitriles (6 -9). Isonitrile (more generally called isocyanide) containing an isocyano group (ϪNϵC) is an isomer of nitrile. Like nitrile, isonitrile is generally highly toxic and produced in nature, probably for self-defense, by various organisms, including bacteria, fungi, marine sponges, etc. (10, 11). For example, xanthocillin was first isolated as an isocyano metabolite from Penicillium notatum, and this isonitrile exhibits a wide antibiotic activity spectrum (12). There have been many other reports about such naturally occurring isocyanides, but information is quite limited on the metabolism of isonitrile. Some metabolic isonitrile intermediates have been elucidated through incorporation experiments, and we initially reported two enzymes involved in isonitrile metabolism. First, we discovered isonitrile hydratase in Pseudomonas putida N19-2, which catalyzes the hydration of an isonitrile to the corresponding N-substituted formamide (R-NH-CHO) (6, 7). It has been approved as a new enzyme by the Nomenclature Committee of t...