Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1

The microbial degradation of nitriles is of interest for bioremediation and green chemistry. We demonstrated that the soil bacterium Rhodococcus sp. RHA1 utilizes a range of nitriles, including acetonitrile, as growth substrates. Proteomic analysis identified 13 proteins that were more abundant in acetonitrile-grown cells, including an aliphatic amidase and a protein with no known homologue. Purification of a nitrile hydratase (NHase) from acetonitrile-grown cells identified the unknown protein as the beta subunit of a two-subunit NHase. Sequence analysis revealed that the genes encoding the amidase (anhC) and the NHase (anhAB) occur in a 12.8 kbp cluster located on plasmid pRHL2. The anh gene cluster also encodes an acetyl-CoA hydrolase, transcriptional regulators, a putative cobalt transporter and a protein of unknown function. Striking features of the NHase include the amino acid sequence identity (32%) and large size (63 and 56 kDa) of the a and beta subunits, as well as the enzyme's metal ion content (one cobalt, two copper and one zinc). The enzyme possessed similar specificities for acetonitrile and propionitrile (k(cat/)Xmr(similar to 7) mM(-1) s(-1)) followed by acrylonitrile and butyronitrile. We propose that this acetonitrile hydratase (ANHase) represents the first member of a previously unknown class of NHases.