title: A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio.
abstract: The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature 415, 497-502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil-borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.
authors.affiliation: Department of Genetics and Microbiology, University of Murcia, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Hernández-Romero
authors.forename: Antonio
authors.initials: A
authors.lastname: Sanchez-Amat
authors.forename: Francisco
authors.initials: F
authors.lastname: Solano
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D005798:Genes, Bacterial; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043368:Ralstonia solanacearum / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase