tailieunhanh - Báo cáo khoa học: On the reaction of D-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity

Evidence is accumulating that oxygen access in proteins is guided and con-trolled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoproteinD-amino acid oxi-dase (DAAO) that have a high affinity for dioxygen and are in close prox-imity to the flavin. | IFEBS Journal On the reaction of D-amino acid oxidase with dioxygen O2 diffusion pathways and enhancement of reactivity Elena Rosini Gianluca Molla Sandro Ghisla and Loredano Pollegioni Dipartimento di Biotecnologie e Scienze Molecolari Universita degli Studi dell Insubria and The Protein Factory Centro Interuniversitario di Biotecnologie Proteiche Politecnico di Milano and Universita degli Studi dell Insubria Varese Italy Keywords flavoproteins mutagenesis oxidases oxygen diffusion oxygen reactivity Correspondence L. Pollegioni Dipartimento di Biotecnologie e Scienze Molecolari University degli Studi dell Insubria Varese Italy Fax 39 332 421500 Tel 39 332 421506 E-mail Received 19 July 2010 revised 2 November 2010 accepted 20 November 2010 doi Evidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase DAAO that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wildtype DAAO with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction but mainly affect the protein conformation and stability. However the T201L variant shows an up to a threefold increase in the rate constant for reaction of O2 with reduced flavin together with a fivefold decrease in the Km for dioxygen. This effect was not observed when a valine is located

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