title: Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase-peroxygenase.
abstract: Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (epsilon (303) = 2,010 M(-1) cm(-1)), and the apparent Michaelis-Menten (K (m)) and catalytic (k (cat)) constants for the reaction were estimated to be 320 microM and 166 s(-1), respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase-peroxygenases.
authors.affiliation: Unit of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, 02763 Zittau, Germany.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Kluge
authors.forename: René
authors.initials: R
authors.lastname: Ullrich
authors.forename: Katrin
authors.initials: K
authors.lastname: Scheibner
authors.forename: Martin
authors.initials: M
authors.lastname: Hofrichter
mesh_terms: D000363:Agaricales / Q000201:enzymology*; D006900:Hydroxylation; D006899:Mixed Function Oxygenases; D009281:Naphthalenes / Q000032:analysis* / Q000378:metabolism; D009284:Naphthols / Q000032:analysis* / Q000378:metabolism; D010544:Peroxidases; D013056:Spectrophotometry, Ultraviolet
chemical_list: D009281:Naphthalenes; D009284:Naphthols; C031721:naphthalene; C029350:1-naphthol; D006899:Mixed Function Oxygenases; D010544:Peroxidases; C022333:peroxygenase