Studies on the cofactors and reaction mechanisms of nitrile hydratase which is useful for the synthesis of amides

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We have established, the enzymatic production process of acrylamide using the microbial nitrile hydratase and the industrial production of acrylamide was started since several years ago. Compared to the high development of the applied works, the basic studies on nitrile hydratase do not progress fully. Thus, the present works focused on more basic works, the elucidation of the cofactors and reaction mechanisms of nitrile hydratase. The following results are summarized.(1) There are two kinds of nitrile hydratases, iron or cobalt is involved as a cofactor.(2) ESR studies showed the following : nitrile hydratase is the first known nonheme iron enzyme with a typical low-spin Fe (III) coordination environment, the axial position of the iron (III) site in the native enzyme may be occupied by thiolate and equo groups, and aliphatic nutrile-substrates directly bind to the iron (III)-active center through water substrate replacement. Cobalt also probably plays the same role, the binding of nitrile and activation of it.(3) Iron nitrile hydratase involves an active carbonyl cofactor as a second prosthetic group, probably pyrrologuinoline quinone. There may be an interaction, such as a hydrogen bond, between the ferric site and a form of hydrated PQQ.(4) The iron and cobalt nitrile hydratases were cloned. The sequence of these genes was determined and the extreme high homology of two genes was found in spite of the difference of confactors.