The role of leucine 191 of Escherichia coli uracil DNA glycosylase in the formation of a highly stable complex with the substrate mimic, ugi, and in uracil excision from the synthetic substrates.
Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic. Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi. In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India.
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Handa
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Roy
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Varshney
D019943:Amino Acid Substitution; D045647:DNA Glycosylases; D004260:DNA Repair; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007364:Intercalating Agents; D007700:Kinetics; D007930:Leucine; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase; D014508:Urea; D014764:Viral Proteins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D014498:Uracil; D014508:Urea; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D007930:Leucine