Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY.
The mutY gene product of Escherichia coli is a 39-kDa protein that catalyzes the removal of adenine bases mispaired with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. Although adenine removal proceeds via monofunctional glycosylase activity, MutY is able to form covalent adducts with substrate DNA in the presence of borohydride, a trait otherwise known to be associated only with enzymes having bifunctional glycosylase/AP lyase activity. To help identify active site residues involved in the formation of MutY-DNA adducts in the presence of borohydride, a series of site-directed mutant forms of MutY were generated. Our data show that Lys 142 is the primary residue involved in cross-link formation. The absence of Lys 142 results in near elimination of the enzyme-DNA adducts formed relative to wild-type, suggesting that this residue is the primary one involved in forming covalent associations with DNA during MutY catalysis. Importantly, the enzymatic activity and DNA binding of the K142A enzyme is nearly identical to the WT enzyme. This shows that formation of the covalent intermediate is not required for adenine removal by MutY. Furthermore, this suggests that the covalent intermediate is formed by reaction of Lys 142 with the OG/G:(AP site) product, and this may be a consequence of MutY's unusually high affinity for the product of its glycosylase action.
Williams
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SD
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
David
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SS
D001894:Borohydrides / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012293:Rifampin / Q000737:chemistry / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D001894:Borohydrides; D018736:DNA Adducts; D012545:Schiff Bases; C025364:sodium borohydride; C009282:sodium cyanoborohydride; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D008239:Lysine; D012293:Rifampin