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The cloning of two open reading frames encoding a heterodimeric protein related in sequence to bacterial sarcosine oxidase and dimethylglycine oxidase from hyperthermophilic Pyrococcus furiosus DSM 3638 is described. the protein was overexpressed in E. coli, purified to homogeneity and identified as a flavoprotein by virtue of the enzymes UV-visible absorption spectrum showing characteristic lambdamax at 367 and 450 nm. The physiological substrate was identified as L-proline (t ½ = ∼105 s-1 for bleaching of flavoprotein spectrum at 450 nm). Additionally, the enzyme oxidises L-pipecolic acid ( t½ = ∼110.5 s-1) and, to a lesser extent sarcosine (t½ = ∼654 s-1). No reactivity with sodium sulfite was detected, consistent with the enzyme belonging to the flavoprotein dehydrogenase class. These data classified the enzyme as a novel hyperthermophilic L-proline dehydrogenase.;The crystal structure of PRODH at 3.3 A resolution shows the enzyme is a heterooctamer (alphabeta)4. The holoenzyme contains one mol each of FAD, FMN and ATP per mol of alphabeta complex. Isolation of monoflavinylated enzyme containing a single FAD cofactor permitted detailed redox potentiometry and pH-dependence studies of the reaction with L-proline. A bell-shaped dependence for kcat/K m as a function of pH was observed with macroscopic pK a values (7.0 +/- 0.2 and 7.6 +/- 0.2) attributed to residue ionisations in the free enzyme. The pH dependence of k cat is sigmoidal, with maximum activity realised in the alkaline region; the dependence is described by a macroscopic pK a value of 7.7 +/- 0.1 and by analogy with other flavoenzymes is tentatively attributed to the ionisation of L-proline in the Michaelis complex. Studies with H225A, H225Q and Y251F mutants ruled out the participation of these residues as catalytic bases.