Active site aspartate residues are critical for tryptophan tryptophylquinone biogenesis in methylamine dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue βTrp⁵⁷ and covalent cross-linking of βTrp⁵⁷ to βTrp¹⁰⁸ to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues βAsp⁷⁶ and βAsp ³² are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative βD76N and βD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. βD76N MADH possessed the six disulfides but had no oxygen incorporated into βTrp⁵⁷ and was completely inactive. The βD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into βTrp⁵⁷ and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the βD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the K_m value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.