title: Understanding quinone cofactor biogenesis in methylamine dehydrogenase through novel cofactor generation.
abstract: Cofactors made from constitutive amino acids in proteins are now known to be relatively common. A number of these involve the generation of quinone cofactors, such as topaquinone in the copper-containing amine oxidases, and lysine tyrosylquinone in lysyl oxidase. The biogenesis of the quinone cofactor tryptophan tryptophylquinone (TTQ) in methylamine dehydrogenase (MADH) involves the post-translational modification of two constitutive Trp residues (Trp(beta)(57) and Trp(beta)(108) in Paracoccus denitrificans MADH). The modifications for generating TTQ are the addition of two oxygens to the indole ring of Trp(beta)(57) and the formation of a covalent cross-link between Cepsilon3 of Trp(beta)(57) and Cdelta1 of Trp(beta)(108). The order in which these events occur is unknown. To investigate the role Trp(beta)(108) may play in this process, this residue was mutated to both a His (betaW108H) and a Cys (betaW108C) residue. For each mutant, the majority of the protein that was isolated was inactive and exhibited weaker subunit-subunit interactions than native MADH. Analysis by mass spectrometry suggested that the inactive protein was a biosynthetic intermediate with only one oxygen atom incorporated into Trp(beta)(57) and no cross-link with residue beta108. However, in each mutant preparation, a small percentage of the mutant enzyme was active and appears to possess a functional tryptophylquinone cofactor. In the case of betaW108C, this cofactor may be identical to cysteine tryptophylquinone, recently described in the bacterial quinohemoprotein amine dehydrogenase. In betaW108H, the active cofactor is presumably a histidine tryptophylquinone, which has not been previously described, and represents the synthesis of a novel quinone protein cofactor.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Arwen R
authors.initials: AR
authors.lastname: Pearson
authors.forename: Limei H
authors.initials: LH
authors.lastname: Jones
authors.forename: LeeAnn
authors.initials: L
authors.lastname: Higgins
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Carrie M
authors.initials: CM
authors.lastname: Wilmot
authors.forename: Victor L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D001483:Base Sequence; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D017931:DNA Primers; C004532:quinone; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors