PURIFICATION AND CHARACTERIZATION OF A NEW GAMMA-GLUTAMYLMETHYLAMIDE-DISSIMILATING ENZYME-SYSTEM FROM METHYLOPHAGA SP AA-30

A gamma-glutamylmethylamide (gamma-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 360 kDa, It consisted of two dissimilar subunits with molecular masses of 55 and 29 kDa, L protein had a molecular mass of 38 kDa 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 gamma-GMA-dissimilating enzyme system catalyzed the formation of glutamate, formaldehyde, 2-ketoglutarate, and ammonia from gamma-GMA, 2-ketoglutarate, and ammonia via the two-step reaction shown below. gamma-GMA --> 2-ketoglutarate + formaldehyde + 2 NH3 2-ketoglutarate + NH3 --> glutamate This conclusion is based on the observations that: 1, The gamma-GMA-dissimilating reaction is catalyzed by both H protein and L protein and requires the presence of IL-ketoglutarate and ammonia. 2, gamma-GMA is consumed with stoichiometric formation of formaldehyde, glutamate, and ammonia in the reaction. 3, Radioactive formaldehyde is formed when [methylamido-C-14]gamma-GMA, but not [glutaryl-U-C-14]gamma-GMA, is added to the reaction mixture as the substrate, Radioactive glutamate is formed from [U-C-14]2-ketoglutarate and radioactive 2-ketoglutarate from [glutaryl-U-C-14]gamma-GMA.