Purification and Characterization of a New γ-Glutamylmethylamide-dissimilating Enzyme System from Methylophaga sp. AA-30

Abstract

A γ-glutamylmethylamide (γ-GMA)-dissimilating enzyme system, H protein and L protein, was purified to homogeneity from a cell-free extract of Methylophaga sp. AA-30. H protein contained flavin and had a molecular mass of 360kDa. It consisted of two dissimilar subunits with molecular masses of 55 and 29kDa. L protein had a molecular mass of 38kDa and contained 1 mol of thiol group per mol of protein. Since L protein in the native form was very labile, the protein was treated with 5, 5'-dithiobis(2-nitrobenzoate) to produce a stable but inactive derivative of the protein, which was purified then reactivated with dithiothreitol. The γ-GMA-dissimilating enzyme system catalyzed the formation of glutamate, formaldehyde, 2-ketoglutarate, and ammonia from γ-GMA, 2-ketoglutarate, and ammonia via the two-step reaction shown below.