Putative secondary active site of bovine pancreatic deoxyribonuclease I.
Previous structural studies based on the co-crystal of a complex between bovine pancreatic deoxyribonuclease I (bpDNase I) and a double-stranded DNA octamer d(GCGATCGC)(2) have suggested the presence of a putative secondary active site near Ser43. In our present study, several crucial amino acid residues postulated in this putative secondary active site, including Thr14, Ser43, and His44 were selected for site-directed mutagenesis. A series of single, double and triple mutants were thus constructed and tested for their DNase I activity by hyperchromicity assay. Substitution of each or both of Thr14 and Ser43 by alanine results in mutant enzymes retaining 30-70% of WT bpDNase I activity. However, when His44 was replaced by aspartic acid, either in the single, double, or triple mutant, the enzyme activities were drastically decreased to 0.5-5% that of WT bpDNase I. Interestingly, when cysteine was substituted for Thr14 or Ser43, the specific DNase activities of the mutant enzymes were substantially increased by 1.5-100-fold, comparing to their alanine substitution mutant counterparts. Two other more sensitive DNase activity assay method, plasmid scission and zymogram analyses further confirm these observations. These results suggested that His44 may play a critical role in substrate DNA binding in this putative secondary active site, and introduction of sulfhydryl groups at Thr14 and Ser43 may facilitate Mn(2+)-coordination and further contribute to the catalytic activity of bpDNase I.
Chen
Wei-Jung
WJ
Graduate Institute of Biotechnology, College of Bioresources, National Ilan University, 1, Shen-Lung Road Sec. 1, Ilan 26047, Taiwan. wjchen@niu.edu.tw
Huang
Po-Tsang
PT
Cheng
Yu-Che
YC
Liao
Ta-Hsiu
TH
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*
D006639:Histidine; D004247:DNA; D003850:Deoxyribonuclease I