Kinetic and structural analysis of bisubstrate inhibition of the Salmonella enterica aminoglycoside 6′-N-acetyltransferase

Maria L B Magalhães, Matthew W. Vetting, Feng Gao, Lee Freiburger, Karine Auclair, John S. Blanchard

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Aminoglycosides are antibacterial compounds that act by binding to the A site of the small 30S bacterial ribosomal subunit and inhibiting protein translation. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including phosphorylation, adenylylation, and acetylation. Bisubstrate analogs for the aminoglycoside N-acetyltransferases are nanomolar inhibitors of Enterococcus faecium AAC(6′)-Ii. However, in the case of the Salmonella enterica aac(6′)-Iy-encoded aminoglycoside N-acetyltransferase, we demonstrate that a series of bisubstrate analogs are only micromolar inhibitors. In contrast to studies with AAC(6′)-Ii, the inhibition constants toward AAC(6′)-Iy are essentially independent of both the identity of the aminoglycoside component of the bisubstrate and the number of carbon atoms that are used to link the CoA and aminoglycoside components. The patterns of inhibition suggest that the CoA portion of the bisubstrate analog can bind to the enzyme-aminoglycoside substrate complex and that the aminoglycoside portion can bind to the enzyme-CoA product complex. However, at the high concentrations of bisubstrate analog used in crystallization experiments, we could crystallize and solve the three-dimensional structure of the enzyme-bisubstrate complex. The structure reveals that both the CoA and aminoglycoside portions bind in essentially the same positions as those previously observed for the enzyme-CoA-ribostamycin complex, with only a modest adjustment to accommodate the "linker". These results are compared to previous studies of the interaction of similar bisubstrate analogs with other aminoglycoside N-acetyltransferases.

Original languageEnglish (US)
Pages (from-to)579-584
Number of pages6
JournalBiochemistry
Volume47
Issue number2
DOIs
StatePublished - Jan 15 2008

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Salmonella
Salmonella enterica
Aminoglycosides
Structural analysis
Coenzyme A
Kinetics
Bacterial Small Ribosome Subunits
Enzymes
Acetyltransferases
Ribostamycin
Enterococcus faecium
Protein Biosynthesis
Acetylation
Crystallization
aminoglycoside N(6')-acetyltransferase
Phosphorylation
Carbon
Anti-Bacterial Agents
Atoms
aminoglycoside acetyltransferase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and structural analysis of bisubstrate inhibition of the Salmonella enterica aminoglycoside 6′-N-acetyltransferase. / Magalhães, Maria L B; Vetting, Matthew W.; Gao, Feng; Freiburger, Lee; Auclair, Karine; Blanchard, John S.

In: Biochemistry, Vol. 47, No. 2, 15.01.2008, p. 579-584.

Research output: Contribution to journalArticle

Magalhães, Maria L B ; Vetting, Matthew W. ; Gao, Feng ; Freiburger, Lee ; Auclair, Karine ; Blanchard, John S. / Kinetic and structural analysis of bisubstrate inhibition of the Salmonella enterica aminoglycoside 6′-N-acetyltransferase. In: Biochemistry. 2008 ; Vol. 47, No. 2. pp. 579-584.
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