Purification, characterization, and crystallization of human pyrroline-5-carboxylate reductase

Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escherichia coli and purified to homogeneity by chromatography. Enzymatic assays of the wild-type protein were carried out using 3,4-dehydro-l-proline as substrate and NAD+ as cofactor. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Human P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 °C. Diffraction data were obtained to a resolution of 2.8 Å and were suitable for high resolution X-ray structure determination.