Functional Expression of Nitrile Hydratase in Escherichia coli:

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dis-sociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase a subunit, NHase 0 subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the y9 subunit gene was co-expressed and the transformant was grown at 30'C or less. A ligand cysteine, aCysll2, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of aCysl l4 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide. Key words: hydration, metalloprotein, nitrile, overexpression, post-translational modi-fication. Nitrile hydratase (NHase; EC 4.2.1.84) catalyzes th