A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family.
We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.
Yasuda
T
T
Division of Medical Genetics and Biochemistry, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan.
Takeshita
H
H
Iida
R
R
Ueki
M
M
Nakajima
T
T
Kaneko
Y
Y
Mogi
K
K
Kominato
Y
Y
Kishi
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D005399:Fishes / Q000235:genetics / Q000378:metabolism; D043143:Hepatopancreas / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010802:Phylogeny; D016415:Sequence Alignment
D018076:DNA, Complementary; D003850:Deoxyribonuclease I