tailieunhanh - Báo cáo khoa học: A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio Role of the seventh histidine and accessibility to the active site

The sequencing of the genome ofRalstonia solanacearum[Salanoubat M, Genin S, Artiguenave F,et al.(2002) Nature415, 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 | ềFEBS Journal A tyrosinase with an abnormally high tyrosine hydroxylase dopa oxidase ratio Role of the seventh histidine and accessibility to the active site Diana Hernandez-Romero1 Antonio Sanchez-Amat1 and Francisco Solano2 1 Department of Genetics and Microbiology 2 Department of Biochemistry and Molecular Biology B University of Murcia Spain Keywords catecholoxidase copper enzymes monophenolase phenol oxidase tyrosinase Correspondence F. Solano Department of Biochemistry and Molecular Biology B Schoolof Medicine University of Murcia Murcia 30100 Spain Fax 34 9683 64150 Tel 34 9683 67194 E-mail address psolano@ URL bbmbi Received 25 July 2005 revised 6 October 2005 accepted 27 October 2005 doi 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