Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site.
Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3' and 5' RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His(211) in this conserved motif was shown to be very important in catalysis by site-specific modification with (14)C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg(2+) and Ca(2+). The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.
Wang
W Y
WY
Institute of Biochemistry, College of Medicine, Taiwan University, 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.
Liaw
S H
SH
Liao
T H
TH
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D033364:Decapoda; D004064:Digestive System / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D060829:Arthropod Proteins; D018076:DNA, Complementary; D004220:Disulfides; D004720:Endonucleases