tailieunhanh - Báo cáo khoa học: Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme

Aryl-alcohol oxidase provides H2O2 for lignin biodegradation, a key pro-cess for carbon recycling in land ecosystems that is also of great biotechno-logical interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. | ễFEBS Journal Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase an H2O2-producing ligninolytic enzyme Patricia Ferreira1 Francisco J. Ruiz-Duenas1 Maria J. Martinez1 Willem J. H. van Berkel2 and Angel T. Martinez1 1 Centro de Investigaciones Biológicas CSIC Madrid Spain 2 Laboratory of Biochemistry Wageningen University Wageningen the Netherlands Keywords aryl-alcoholoxidase EC flavoenzyme molecular docking site-directed mutagenesis substrate-binding site Correspondence A. T. Martinez Centro de Investigaciones Biologicas CSIC Ramiro de Maeztu 9 E-28040 Madrid Spain Fax 34 915360432 Tel 34 918373112 E-mail ATMartinez@ Present address Department of Biochemistry and Molecular Biology College of Medicine DrexelUniver-sity Philadelphia PA USA Received 17 July 2006 revised 26 August 2006 accepted 1 September 2006 doi Aryl-alcohol oxidase provides H2O2 for lignin biodegradation a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans and characterized in terms of their steady-state kinetic properties. Two histidine residues His502 and His546 are strictly required for aryl-alcohol oxidase catalysis as shown by the lack of activity of different variants. This fact together with their location near the isoalloxazine ring of FAD suggested a contribution to catalysis by alcohol activation enabling its oxidation by flavin-adenine .

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