Discovery of a novel enzyme, isonitrile hydratase, involved in nitrogen-carbon triple bond cleavage

Isonitrile containing an N equivalent toC triple bond was degraded by microorganism sp N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide tan isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K-m value for cyclohexyl isocyanide mas 16.2 mM. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.