Bioinformatics of a novel nitrile hydratase gene cluster of the N2-fixing bacterium Microvirga flocculans CGMCC 1.16731 and characterization of the enzyme.

Microvirga flocculans CGMCC 1.16731 can degrade many cyano group-containing neonicotinoid insecticides. Here, its genome was sequenced, and a novel nitrile hydratase gene cluster was discovered in a plasmid. The NHase gene cluster (pnhF) has gene structure beta-subunit 1, alpha-subunit, and beta-subunit 2, which is different from previously reported NHase gene structures. Phylogenetic analysis of alpha-subunits indicated that NHases containing the three subunit (beta 1 alpha beta 2) structure are independent from NHases containing two subunits (alpha beta). pnhF was successfully expressed in Escherichia coli, and the purified PnhF could convert the nitrile-containing insecticide flonicamid to N-(4-trifluoromethylnicotinoyl)glycinamide. The enzymatic properties of PnhF were investigated using flonicamid as a substrate. Homology models revealed that amino acid residue beta 1-Glu56 may strongly affect the catalytic activity of PnhF. This study expands our understanding of the structures and functions of NHases and the enzymatic mechanism of the environmental fate of flonicamid.