title: Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis.
abstract: The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, Florida 32827, United States.
authors.forename: Heather R
authors.identifier: 0000-0002-2413-659X
authors.initials: HR
authors.lastname: Williamson
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, Florida 32827, United States.
authors.forename: Esha
authors.initials: E
authors.lastname: Sehanobish
authors.affiliation: Division of Marine Science, The University of Southern Mississippi, Stennis Space Center , Mississippi 39529, United States.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Shiller
authors.affiliation: Department of Genetics and Microbiology, University of Murcia , Murcia 30100, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Sanchez-Amat
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida , Orlando, Florida 32827, United States.
authors.forename: Victor L
authors.identifier: 0000-0002-1966-7302
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D045563:Indolequinones / Q000737:chemistry / Q000378:metabolism*; D053544:Marinomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D045563:Indolequinones; D011994:Recombinant Proteins; C447619:cysteine tryptophylquinone; D001224:Aspartic Acid; D003300:Copper; D014364:Tryptophan; D000594:Amino Acid Oxidoreductases