Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I

被引:15
作者
Evans, SJ [1 ]
Shipstone, EJ [1 ]
Maughan, WN [1 ]
Connolly, BA [1 ]
机构
[1] Newcastle Univ, Dept Biochem & Genet, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England
关键词
D O I
10.1021/bi9824893
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:3902 / 3909
页数:8
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共 54 条
[1]  
BOIX E, 1994, J BIOL CHEM, V269, P2529
[2]   NUCLEAR MAGNETIC-RESONANCE AND NEUTRON-DIFFRACTION STUDIES OF THE COMPLEX OF RIBONUCLEASE-A WITH URIDINE VANADATE, A TRANSITION-STATE ANALOG [J].
BORAH, B ;
CHEN, CW ;
EGAN, W ;
MILLER, M ;
WLODAWER, A ;
COHEN, JS .
BIOCHEMISTRY, 1985, 24 (08) :2058-2067
[3]   SEQUENCE-DEPENDENT STRUCTURAL VARIATIONS OF DNA REVEALED BY DNASE-I [J].
BRUKNER, I ;
JURUKOVSKI, V ;
SAVIC, A .
NUCLEIC ACIDS RESEARCH, 1990, 18 (04) :891-894
[4]   Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli:: purification and characterization of the recombinant enzyme [J].
Chen, CY ;
Lu, SC ;
Liao, TH .
GENE, 1998, 206 (02) :181-184
[5]   ENGINEERING RIBONUCLEASE-A - PRODUCTION, PURIFICATION AND CHARACTERIZATION OF WILD-TYPE ENZYME AND MUTANTS AT GLN11 [J].
DELCARDAYRE, SB ;
RIBO, M ;
YOKEL, EM ;
QUIRK, DJ ;
RUTTER, WJ ;
RAINES, RT .
PROTEIN ENGINEERING, 1995, 8 (03) :261-273
[6]   MUTAGENESIS OF THE DNA-BINDING RESIDUES IN BOVINE PANCREATIC DNASE-1 - AN INVESTIGATION INTO THE MECHANISM OF SEQUENCE DISCRIMINATION BY A SEQUENCE SELECTIVE NUCLEASE [J].
DOHERTY, AJ ;
WORRALL, AF ;
CONNOLLY, BA .
NUCLEIC ACIDS RESEARCH, 1991, 19 (22) :6129-6132
[7]   OVERPRODUCTION OF THE TOXIC PROTEIN, BOVINE PANCREATIC DNASEI, IN ESCHERICHIA-COLI USING A TIGHTLY CONTROLLED T7-PROMOTER-BASED VECTOR [J].
DOHERTY, AJ ;
CONNOLLY, BA ;
WORRALL, AF .
GENE, 1993, 136 (1-2) :337-340
[8]   THE ROLES OF ARGININE-41 AND TYROSINE-76 IN THE COUPLING OF DNA RECOGNITION TO PHOSPHODIESTER BOND-CLEAVAGE BY DNASE-I - A STUDY USING SITE-DIRECTED MUTAGENESIS [J].
DOHERTY, AJ ;
WORRALL, AF ;
CONNOLLY, BA .
JOURNAL OF MOLECULAR BIOLOGY, 1995, 251 (03) :366-377
[9]   STRUCTURAL JUNCTIONS IN DNA - THE INFLUENCE OF FLANKING SEQUENCE ON NUCLEASE DIGESTION SPECIFICITIES [J].
DREW, HR ;
TRAVERS, AA .
NUCLEIC ACIDS RESEARCH, 1985, 13 (12) :4445-4467
[10]   DNA STRUCTURAL VARIATIONS IN THE ESCHERICHIA-COLI TYRT-PROMOTER [J].
DREW, HR ;
TRAVERS, AA .
CELL, 1984, 37 (02) :491-502