Aldoxime Dehydratase Co-existing with Nitrile Hydratase and Amidase in the Iron-Type Nitrile Hydratase-Producer Rhodococcus sp. N-771

Abstract

We identified an aldoxime dehydratase (Oxd) gene in the 5′-flanking region of the nitrile hydratase–amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodococcus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A-4, Pseudomonas chlororaphis B23, and Bacillus sp. OxB-1, respectively. The enzyme was expressed in Escherichia coli under the control of the lac- or T7 promoters in its intact and His6-tagged forms, purified, and characterized. The enzyme had heme b as a prosthetic group, catalyzed a stoichiometric dehydration of aldoxime into nitrile, and exhibited the highest activity at neutral pH and at around 30°C similar to the known Oxd from Bacillus sp. OxB-1. The activity was enhanced by reducing agents, such as Na2S, Na2S2O4, 2-mercaptoethanol, and L-cysteine and supplementary additions of electron acceptors such as flavins, sulfite ion, and vitamin K3. The effect of various chemicals on the enzyme activity was different in the presence and absence of the reducing reagent, Na2S. The enzyme preferentially acts on aliphatic-type substrates and the substrate specificity of the enzyme coincides with that reported for nitrile hydratase produced by the strain.