@article {5412, title = {Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus.}, journal = {Protein Expression and Purification}, volume = {64}, year = {2009}, month = {2009 Apr}, pages = {125-30}, abstract = {

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.

}, keywords = {Archaeal Proteins, Chromatography, Crystallization, Crystallography, Gel, Kinetics, NADP, Pyrroline Carboxylate Reductases, Sulfolobus solfataricus, Temperature, Thiazolidines, X-Ray}, issn = {1096-0279}, doi = {10.1016/j.pep.2008.10.018}, author = {Meng, Zhaohui and Liu, Zhe and Lou, Zhiyong and Gong, Xiaocui and Cao, Yi and Bartlam, Mark and Zhang, Keqin and Rao, Zihe} } @article {5416, title = {Crystal structure of human pyrroline-5-carboxylate reductase.}, journal = {Journal of Molecular Biology}, volume = {359}, year = {2006}, month = {2006 Jun 23}, pages = {1364-77}, abstract = {

Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline.

}, keywords = {Amino Acid Sequence, Binding Sites, Dimerization, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Protein Structure, Pyrroline Carboxylate Reductases, Secondary, Sequence Alignment, Streptococcus pyogenes}, issn = {0022-2836}, doi = {10.1016/j.jmb.2006.04.053}, author = {Meng, Zhaohui and Lou, Zhiyong and Liu, Zhe and Li, Ming and Zhao, Xiaodong and Bartlam, Mark and Rao, Zihe} } @article {5414, title = {Purification, characterization, and crystallization of human pyrroline-5-carboxylate reductase.}, journal = {Protein Expression and Purification}, volume = {49}, year = {2006}, month = {2006 Sep}, pages = {83-7}, abstract = {

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.

}, keywords = {Crystallization, Crystallography, Gene Expression, Humans, Polymers, Proline, Pyrroline Carboxylate Reductases, Solubility, Water, X-Ray}, issn = {1046-5928}, doi = {10.1016/j.pep.2006.02.019}, author = {Meng, Zhaohui and Lou, Zhiyong and Liu, Zhe and Hui, Dong and Bartlam, Mark and Rao, Zihe} } @article {5415, title = {Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {103}, year = {2006}, month = {2006 Jun 13}, pages = {8965-70}, abstract = {

Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.

}, keywords = {Animals, Apoptosis, Ascorbic Acid, Crystallography, Cytochromes c, DNA Damage, Electron Transport Complex IV, Erythrocytes, Humans, Hydrogen Peroxide, Male, Mice, Models, Molecular, Oocytes, Oxidants, Phylogeny, Protein Conformation, Protein Isoforms, Reactive Oxygen Species, Spermatozoa, Testis, Water, X-Ray, Xenopus laevis}, issn = {0027-8424}, doi = {10.1073/pnas.0603327103}, author = {Liu, Zhe and Lin, Hao and Ye, Sheng and Liu, Qin-Ying and Meng, Zhaohui and Zhang, Chuan-Mao and Xia, Yongjing and Margoliash, Emanuel and Rao, Zihe and Liu, Xiang-Jun} }