TAILIEUCHUNG - Báo cáo khoa học: Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase⁄oxidase

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary aminesl-proline, l-pipecolic acid and sarcosine, with optimal catalytic activity towards l-proline. | ễFEBS Journal Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase oxidase Phillip J. Monaghan David Leys and Nigel S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester UK Keywords amine oxidation flavoprotein hyperthermophile mechanism proline dehydrogenase Correspondence N. S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester 131 Princess Street Manchester M1 7DN UK Fax 44 161 275 5586 Tel 44 161 275 5632 E-mail Received 4 January 2007 revised 9 February 2007 accepted 19 February 2007 doi Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli purified and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline L-pipecolic acid and sarcosine with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD FMN and ATP per ab complex is not reduced by sulfite and reoxidizes slowly following reduction which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline for which a bell-shaped dependence pKa values and for kcat Km as a function of pH was observed. The pH dependence of kcat is sigmoidal described by a single macroscopic pKa of tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• Crystal structure
• medical analysis
• biochemical studies
• medical knowledge
• medical research
• analysis of cytoplasmic
• oxidation
• bacteria
• hemoglobin
• medicine
• cell proliferation
• breast cancer
• chemical reaction
• gastric cancer
• hormone medicine
• tumor cells
• environmental medicine