| Abstract
| - Cofactors made from constitutive amino acids in proteins are now known to be relativelycommon. A number of these involve the generation of quinone cofactors, such as topaquinone in thecopper-containing amine oxidases, and lysine tyrosylquinone in lysyl oxidase. The biogenesis of the quinonecofactor tryptophan tryptophylquinone (TTQ) in methylamine dehydrogenase (MADH) involves the post-translational modification of two constitutive Trp residues (Trpβ57 and Trpβ108 in Paracoccus denitrificansMADH). The modifications for generating TTQ are the addition of two oxygens to the indole ring ofTrpβ57 and the formation of a covalent cross-link between Cε3 of Trpβ57 and Cδ1 of Trpβ108. The orderin which these events occur is unknown. To investigate the role Trpβ108 may play in this process, thisresidue was mutated to both a His (βW108H) and a Cys (βW108C) residue. For each mutant, the majorityof the protein that was isolated was inactive and exhibited weaker subunit−subunit interactions thannative MADH. Analysis by mass spectrometry suggested that the inactive protein was a biosyntheticintermediate with only one oxygen atom incorporated into Trpβ57 and no cross-link with residue β108.However, in each mutant preparation, a small percentage of the mutant enzyme was active and appearsto possess a functional tryptophylquinone cofactor. In the case of βW108C, this cofactor may be identicalto cysteine tryptophylquinone, recently described in the bacterial quinohemoprotein amine dehydrogenase.In βW108H, the active cofactor is presumably a histidine tryptophylquinone, which has not been previouslydescribed, and represents the synthesis of a novel quinone protein cofactor.
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