The role of two conserved amino acids, glutamine 90 and asparagine 137, in O6-methylguanine-DNA methyltransferase stability, activity and substrate specificity

Russell O. Pieper, Susan E. Morgan, Mark Kelley

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (>50% of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5% and 0.8% of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.

Original languageEnglish (US)
Pages (from-to)1895-1902
Number of pages8
JournalCarcinogenesis
Volume15
Issue number9
StatePublished - Sep 1994
Externally publishedYes

Fingerprint

Asparagine
Methyltransferases
Substrate Specificity
Glutamine
Specificity
Amino Acids
Amino acids
DNA
Substrate
Proteins
Protein
Mutant Proteins
Mutant
Substrates
Repair
Alkyl and Aryl Transferases
Bacteria
O-(6)-methylguanine
Protein Stability
Glutathione Transferase

ASJC Scopus subject areas

  • Cancer Research
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Physiology
  • Behavioral Neuroscience

Cite this

The role of two conserved amino acids, glutamine 90 and asparagine 137, in O6-methylguanine-DNA methyltransferase stability, activity and substrate specificity. / Pieper, Russell O.; Morgan, Susan E.; Kelley, Mark.

In: Carcinogenesis, Vol. 15, No. 9, 09.1994, p. 1895-1902.

Research output: Contribution to journalArticle

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abstract = "To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (>50{\%} of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5{\%} and 0.8{\%} of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.",
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