The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue ␤Trp 57 and covalent cross-linking of ␤Trp 57 to ␤Trp 108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues ␤Asp 76 and ␤Asp 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 ␤D76N and ␤D32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. ␤D76N MADH possessed the six disulfides but had no oxygen incorporated into ␤Trp 57 and was completely inactive. The ␤D32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into ␤Trp 57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the ␤D32N 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.Protein-derived cofactors are catalytic and redox-active prosthetic groups that are derived from posttranslational modification of peptide amino acid residues (1-3). These include covalently cross-linked amino acid residues, stable free radicals derived from amino acid residues, and quinones derived from tyrosine and tryptophan residues. The protein-derived cofactor tryptophan tryptophylquinone (TTQ) 1 (4) is the prosthetic group of methylamine dehydrogenase (MADH) (Fig. 1) (5). MADH is a periplasmic enzyme that catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia and the subsequent electron transfer to a type I copper protein, amicyanin (6). MADH from Paracoccus denitrificans is composed of four subunits that are arranged in a symmetrical ␣ 2 ␤ 2 structure (7). A TTQ is located in each ␤ subunit and is formed by posttranslational modifications of ␤Trp 57 and ␤Trp 108 . As can be seen from its structure (Fig. 1), TTQ biosynthesis requires insertion of oxygens at the C-6 and C-7 positions of the indole ring of ␤Trp 57 and formation of a covalent bond between the indole rings of ␤Trp 57 and ␤Trp 108 . TTQ has also been identified as the prosthetic group of aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis (8). As in MADH, the TTQ is located in the smaller ␤ subunit of AADH, which is 60% identical to the MADH ␤ subunit (9). In addition to TTQ, the MADH ␤ subunit exhibits another unusual structural feature. Of the 131 amino acid residues of the ␤ subunit, there are 12 cysteines, all of which are conserved in MADH from other organisms and in AADH and which form six intrasubunit disulfide bonds (7)...

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