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 amines l-proline, l-pipecolic acid and sarcosine, with optimal catalytic activity towards l-proline. The holoenzyme contains one FAD, FMN and ATP per αβ 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 7.0 ± 0.2 and 7.6 ± 0.2) for kcat/Km as a function of pH was observed. The pH dependence of kcat is sigmoidal, described by a single macroscopic pKa of 7.7 ± 0.1, 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 potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 ± 0.05 mV·C°-1), and extrapolation to physiologic growth temperature for P. furiosus (100°C) yields a value of -407 ± 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic l-proline dehydrogenase. Implications for its mechanism of action are discussed. © 2007 The Authors.