Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

Nature recycles l ‐proline by converting it to l ‐glutamate. This four‐electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ 1 ‐pyrroline‐5‐carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n ‐octyl β‐ d ‐glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P 2 1 2 1 2 1 , with unit‐cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular‐replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino‐acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.