Date of this Version
Biochemistry. 2012 December 18; 51(50): pp. 10099–10108. doi:10.1021/bi301312f.
Proline dehydrogenase catalyzes the FAD-dependent oxidation of proline to Δ1- pyrroline-5- carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analog L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analog binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by the α8 helix and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 Å, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.