title: Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.
abstract: Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok 10900, Thailand.
authors.forename: Penporn
authors.initials: P
authors.lastname: Sujiwattanarat
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok 10900, Thailand.
authors.forename: Parinya
authors.initials: P
authors.lastname: Pongsanarakul
authors.affiliation: Sriracha Moda Co., Ltd., Sriracha, Chonburi 20110, Thailand.
authors.forename: Yosapong
authors.initials: Y
authors.lastname: Temsiripong
authors.affiliation: Sriracha Moda Co., Ltd., Sriracha, Chonburi 20110, Thailand.
authors.forename: Theeranan
authors.initials: T
authors.lastname: Temsiripong
authors.affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Charin
authors.initials: C
authors.lastname: Thawornkuno
authors.affiliation: Laboratory of Animal Genetics, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Uno
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok 10900, Thailand.
authors.forename: Sasimanas
authors.initials: S
authors.lastname: Unajak
authors.affiliation: Laboratory of Animal Genetics, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Matsuda
authors.affiliation: Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok 10900, Thailand; Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU, Thailand), Kasetsart University, Bangkok 10900, Thailand. Electronic address: fsciktc@ku.ac.th.
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
authors.affiliation: Laboratory of Animal Genetics, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University (CASTNAR, NRU-KU, Thailand), Kasetsart University, Bangkok 10900, Thailand; Laboratory of Animal Cytogenetics and Comparative Genomics, Department of Genetics, Faculty of Science, Kasetsart University, 50 Ngamwongwan, Chatuchak, Bangkok 10900, Thailand. Electronic address: kornsorn.s@ku.ac.th.
authors.forename: Kornsorn
authors.initials: K
authors.lastname: Srikulnath
mesh_terms: D000489:Alligators and Crocodiles / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013482:Superoxide Dismutase
keywords: Antioxidant enzyme; Chromosome; Crocodile; Enzyme activity; Superoxide dismutase; mRNA expression