Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity.
We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.
Tang
D
D
Department of Chemistry, Saga Medical School, Saga City 849-8501, Japan.
Ando
S
S
Takasaki
Y
Y
Tadano
J
J
D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D004252:DNA Mutational Analysis / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D015247:Deoxyribonuclease HindIII / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics
D002413:Cations, Divalent; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D015247:Deoxyribonuclease HindIII; D008239:Lysine