The molecular basis of the expansive substrate specificity of the antibiotic resistance enzyme aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2′: The role of Asp-99 as an active site base important for acetyl transfer

David D. Boehr, Stephen I. Jenkins, Gerard D. Wright

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19 Citations (Scopus)

Abstract

The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2″ (AAC(6′). aminoglycoside phosphotransferase-2″, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6′)-Ie, is the only member of the large AAC (6′) subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6′)-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6′)-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.

Original languageEnglish (US)
Pages (from-to)12873-12880
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number15
DOIs
StatePublished - Apr 11 2003

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Microbial Drug Resistance
Substrate Specificity
Catalytic Domain
Aminoglycosides
Anti-Bacterial Agents
Substrates
Enzymes
Acetylation
Mutagenesis
Acetyltransferases
Pathogens
Site-Directed Mutagenesis
Isotopes
Phosphotransferases
Molecules
6'-aminoglycoside acetyltransferase-2''-aminoglycoside phosphotransferase

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "The molecular basis of the expansive substrate specificity of the antibiotic resistance enzyme aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2′: The role of Asp-99 as an active site base important for acetyl transfer",
abstract = "The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2″ (AAC(6′). aminoglycoside phosphotransferase-2″, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6′)-Ie, is the only member of the large AAC (6′) subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6′)-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6′)-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.",
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AU - Jenkins, Stephen I.

AU - Wright, Gerard D.

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N2 - The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2″ (AAC(6′). aminoglycoside phosphotransferase-2″, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6′)-Ie, is the only member of the large AAC (6′) subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6′)-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6′)-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.

AB - The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6′-aminoglycoside phosphotransferase-2″ (AAC(6′). aminoglycoside phosphotransferase-2″, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6′)-Ie, is the only member of the large AAC (6′) subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6′)-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6′)-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.

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