Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.
Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.
Shuman
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Program in Molecular Biology, Sloan-Kettering Institute, New York, NY 10021.
Kane
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Morham
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D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D011994:Recombinant Proteins / Q000378:metabolism; D012440:Saccharomyces / Q000201:enzymology; D014443:Tyrosine; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics*
D003062:Codon; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D014443:Tyrosine; D010649:Phenylalanine; D004264:DNA Topoisomerases, Type I