TAILIEUCHUNG - Báo cáo khoa học: Molecular evolution of shark and other vertebrate DNases I

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicususing three-step column chromatography. Although its enzymatic properties resem-bled those of other vertebrateDNases I, sharkDNase I was unique encoding theDNases I of two shark species,H. japonicusandTriakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of twoCys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca 2+ -binding site. . | Eur. J. Biochem. 271 4428-4435 2004 FEBS 2004 doi Molecular evolution of shark and other vertebrate DNases I Toshihiro Yasuda1 Reiko Iida2. Misuzu Ueki1. Yoshihiko Kominato3 Tamiko Nakajima3 Haruo Takeshita3 Takanori Kobayashi4 and Koichiro Kishi3 1 Division of Medical Genetics and Biochemistry and 2Division of Legal Medicine Faculty of Medical Sciences University of Fukui Japan 3Department of Legal Medicine Gunma University Graduate School of Medicine Japan 4National Research Institute of Fisheries Science Japan We purified pancreatic deoxyribonuclease I DNase I from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species H. japonicus and Triakis scyllia were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes substitution of two Cys residues at positions 101 and 104 which are well conserved in all other vertebrate DNases I and insertion of an additional Thr or Asn residue into an essential Ca2 -binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I which has a high a-helical structure content was replaced with its amphibian fish and shark counterparts which have low a-helical structure contents the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wildtype enzyme. In contrast substitution of the human signal sequence region into the amphibian fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through .

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