Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I.
Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cleaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleotides have revealed interactions between the side chains of several amino acids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The effects these interactions have on enzyme catalysis and DNA hydrolysis selectivity have been investigated by site-directed mutagenesis. Mutations to R111, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interacts with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the correct orientation for efficient hydrolysis. Both T207 and Y211 bind to the phosphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the scissile phosphate and appears to play a direct role in the catalytic mechanism. The mutation N74D, which interacts with a phosphate twice removed from the scissile group, strongly reduced DNA hydrolysis. However, a comparison of DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bonding), are tolerated. S206, which binds to a DNA phosphate two positions away from the cleavage site, appears to play a relatively unimportant role. None of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substrates.
Evans
S J
SJ
Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne, U.K.
Shipstone
E J
EJ
Maughan
W N
WN
Connolly
B A
BA
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000737:chemistry* / Q000235:genetics; D013912:Threonine / Q000737:chemistry / Q000235:genetics; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D012365:RNA, Transfer, Tyr; D013403:Sugar Phosphates; C117362:deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate); D013912:Threonine; D014443:Tyrosine; D012694:Serine; D001216:Asparagine; D004247:DNA; D001120:Arginine; D003850:Deoxyribonuclease I; D013936:Thymidine