Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus.
The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)+. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 degrees C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 3.5A and were suitable for X-ray structure determination.
Meng
Zhaohui
Z
Laboratory of Molecular Cardiology, Department of Cardiology, The First Affiliated Hospital of Kunming Medical College, 295 Xichang Road, Kunming, Yunnan 650032, China. mzhxyh@yahoo.com
Liu
Zhe
Z
Lou
Zhiyong
Z
Gong
Xiaocui
X
Cao
Yi
Y
Bartlam
Mark
M
Zhang
Keqin
K
Rao
Zihe
Z
D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D007700:Kinetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011762:Pyrroline Carboxylate Reductases / Q000737:chemistry* / Q000302:isolation & purification*; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000378:metabolism; D013696:Temperature; D053778:Thiazolidines / Q000737:chemistry; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase
D019843:Archaeal Proteins; D009249:NADP; D011762:Pyrroline Carboxylate Reductases; D053778:Thiazolidines; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase; C003438:thiazolidine-4-carboxylic acid