tailieunhanh - Báo cáo khoa học: Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Tham khảo luận văn - đề án 'báo cáo khoa học: covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process', luận văn - báo cáo, báo cáo khoa học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | ỊFEBS Journal Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process Jianfeng Jin1 Hortense Mazon2 Robert H. H. van den Heuvel2 f Albert J. Heck2 Dick B. Janssen1 and Marco W. Fraaije1 1 Laboratory of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen The Netherlands 2 Biomolecular Mass Spectrometry and Proteomics Group Bijvoet Center for Biomolecular Research and Utrecht Institute for PharmaceuticalSciences Utrecht University The Netherlands Keywords covalent flavinylation FAD post-translational modification tandem ESI-MS vanillyl-alcohol oxidase Correspondence M. W. Fraaije Laboratory of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Nijenborgh 4 9747 AG Groningen The Netherlands Fax 31 50 3634165 Tel 31 50 3634345 E-mail Present address Nancy-Universite MAEM CNRS UMR 7567 Faculte des Sciences Vandoeuvre France ỊNV Organon a part of Schering-Plough Corporation BH Oss The Netherlands Received 23 June 2008 revised 12 August 2008 accepted 18 August 2008 doi Vanillyl-alcohol oxidase VAO EC contains a covalently 8a-histidyl bound FAD which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration displaying a hyperbolic relationship KFAD M kactivation min-1 . The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD as determined by SDS PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process which proceeds via a reduced flavin intermediate. .