Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I.
Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase-DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Daniel
D
Strahs
Chang-Xi
CX
Zhu
Bokun
B
Cheng
Jason
J
Chen
Yuk-Ching
YC
Tse-Dinh
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D019295:Computational Biology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; D012694:Serine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008239:Lysine