Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

Abstract

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...

Heterologous expression, purification and characterization of arylacetonitrilases fromNectria haematococcaandArthroderma benhamiae

“…An insert of the same length was also found in almost all the aforementioned hypothetical nitrilases homologous with NitNc except for the hypothetical protein from Trichoderma virens . Of the other hypothetical nitrilases with lower amino acid sequence identities, only those from the yeast, Meyerozyma guilliermondi and the bacteria Saccharomonospora viridis, and Stappia aggregata (with 38 -42% identity to the N. crassa nitrilase) contained a similar sequence at the corresponding position, which was also found in the isonitrile hydratase from Arthrobacter pascens (Sato et al 2010) with 42% identity to NitNc. The importance of this insert to the catalytic properties of the enzymes remains to be clarifi ed.…”

A New Synthetic Route to N -Benzyl Carboxamides through the Reverse Reaction of N -Substituted Formamide Deformylase

“…We are interested in how C-N hydrolases evolved (27,49,50). Because N-substituted formamide contains a nitrogen-carbon bond, an N-substituted formamide deformylase belongs to the C-N hydrolases.…”

“…Isonitrile hydratase and N-substituted formamide deformylase have been approved as new enzymes by the International Union of Biochemistry and Molecular Biology (IUBMB), and new EC numbers (EC 4.2.1.103 and EC 3.5.1.91, respectively) have been given to them. Very recently, we discovered the second isonitrile hydratase, which cooperated with N-substituted formamide deformylase in Arthrobacter pascens F164 (27). These two isonitrile hydratases are biochemically, immunologically, and structurally different from each other.…”

“…These two isonitrile hydratases are biochemically, immunologically, and structurally different from each other. However, there have been only three reports (24)(25)(26)(27) on the enzymes involved in isonitrile metabolism.…”

Complete Genome Sequencing of Protease-Producing Novel Arthrobacter sp. Strain IHBB 11108 Using PacBio Single-Molecule Real-Time Sequencing Technology

“…More than 84 species have been described from alpine, Antarctic, forest, field, and rhizosphere soils; Antarctic lake and deep-sea sediments; South China Sea and Pohang basin waters; and extreme environments, including air in the Russian space laboratory, alpine glacier cryoconite, and an alpine ice cave ( http://www.bacterio.net/arthrobacter.html ). Members of this genus are involved in the biodegradation of the plant alkaloids nicotine ( 4 ), 2,4-dinitrotoluene ( 5 ), and 4-fluorocinnamic acid ( 6 ), and in the production of the enzymes isonitrile hydratase ( 7 ), serine hydroxymethyltransferase ( 8 ), beta-galactosidase ( 9 ), and dehydrogenases ( 10 ). During exploration of microbial communities from sediment and water samples collected from Chandra Tal Lake (32.29°N 77.36°E), an off-white, psychrotrophic isolate showing hydrolysis of skimmed milk was characterized as a novel species based on the highest identity of 97.46% from a 16S rRNA gene sequence comparison with Arthrobacter russicus GTC 863 (T) .…”