Meng Zhaohui, Lou Zhiyong, Liu Zhe, Hui Dong, Bartlam Mark, Rao Zihe
Tsinghua-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing 100084, China.
Protein Expr Purif. 2006 Sep;49(1):83-7. doi: 10.1016/j.pep.2006.02.019. Epub 2006 Mar 20.
Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-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 degrees C. Diffraction data were obtained to a resolution of 2.8A and were suitable for high resolution X-ray structure determination.
吡咯啉-5-羧酸还原酶（P5CR）催化Δ1-吡咯啉-5-羧酸（P5C）还原为脯氨酸，同时将NAD（P）H氧化为NAD（P）⁺。P5C和脯氨酸之间的酶促循环在许多生理和病理过程中非常重要。人P5CR在大肠杆菌中过表达，并通过色谱法纯化至同质。使用3,4-脱氢-L-脯氨酸作为底物和NAD⁺作为辅因子对野生型蛋白进行酶活性测定。通过交联和尺寸排阻凝胶过滤色谱对均聚物进行表征。人P5CR在37℃下通过悬滴气相扩散法结晶。获得了分辨率为2.8Å的衍射数据，适用于高分辨率X射线结构测定。
