Contrasting effects of mutating active site residues, aspartic acid 64 and histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein.
Uracil, a promutagenic base, arises in DNA by spontaneous deamination of cytosine or by the malfunctioning of DNA polymerases. To maintain the genomic integrity, cells possess a highly conserved base excision repair enzyme, uracil-DNA glycosylase (UDG). UDGs have a notably high turnover number and strict specificity for uracil in DNA. UDGs are inhibited by a small proteinaceous inhibitor, Ugi, which acts as a transition state substrate mimic. Crystal structure studies have identified the residues crucial in catalysis, and in their interaction with Ugi. Here, we report on the mutational analyses of D64 (D64H and D64N) and H187 (H187C, H187L and H187R) in the active site pocket of Escherichia coli UDG. The mutants were compromised in uracil excision by approximately 200-25,000 fold when compared to the native protein. In contrast, our analysis of the in vivo formed UDG-Ugi complexes on urea gels shows that D64 and H187 contribute minimally to the interaction of the two proteins. Thus, our findings provide further evidence to the primary function of D64 and H187 in catalysis.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Priya
P
Handa
Narottam
N
Acharya
Ramappa K
RK
Talawar
Sudipta
S
Roy
Umesh
U
Varshney
D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D008957:Models, Genetic; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D014498:Uracil / Q000737:chemistry*; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009841:Oligonucleotides; D001224:Aspartic Acid; D006639:Histidine; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase