Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I.
The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.
Cheng
Bokun
B
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Sorokin
Elena P
EP
Tse-Dinh
Yuk-Ching
YC
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005998:Glycine / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D008715:Methionine / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D010641:Phenotype; D013014:SOS Response, Genetics; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry; D014633:Valine / Q000235:genetics; D015010:Yersinia pestis / Q000201:enzymology
D014443:Tyrosine; D012694:Serine; D004247:DNA; D008715:Methionine; D004264:DNA Topoisomerases, Type I; D014633:Valine; D008274:Magnesium; D005998:Glycine