A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity.
The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.
Williams
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Department of Chemistry, University of Utah, Salt Lake City, Utah 84112-0850, USA.
David
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D000595:Amino Acid Sequence; D001894:Borohydrides / Q000494:pharmacology; D019757:Carbon-Oxygen Lyases / Q000145:classification / Q000235:genetics*; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003839:Deoxyadenosines / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005573:Formycins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000145:classification / Q000235:genetics*; D017354:Point Mutation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001894:Borohydrides; D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005573:Formycins; D009097:Multienzyme Complexes; C047923:2'-deoxyaristeromycin; C030964:2'-fluoro-2'-deoxyadenosine; C025364:sodium borohydride; C045970:2'-deoxyformycin A; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D006026:Glycoside Hydrolases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C058118:2'-deoxyadenosine