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Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and
G A McKay1, P R Thompson, G D Wright
1Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Biochemistry
|June 7, 1994
Summary
Aminoglycoside phosphotransferases (APHs) inactivate antibiotics. This study characterizes enterococcal APH(3')-IIIa, revealing its broad-spectrum inactivation mechanism and key structural features for antibiotic resistance.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Aminoglycoside phosphotransferases (APHs) confer bacterial resistance to essential antibiotics.
- Understanding APH enzyme mechanisms is crucial for combating antibiotic resistance.
Purpose of the Study:
- To characterize the enterococcal enzyme APH(3 ')-IIIa.
- To elucidate the mechanism of aminoglycoside inactivation by APH(3 ')-IIIa.
- To identify structure-activity relationships for designing novel inhibitors.
Main Methods:
- Protein overproduction and purification of APH(3 ')-IIIa.
- Enzyme kinetics (Km, kcat) and substrate inhibition studies.
- Nuclear Magnetic Resonance (NMR) spectroscopy for structural analysis.
Main Results:
- APH(3 ')-IIIa inactivates a broad spectrum of aminoglycosides via ATP-dependent O-phosphorylation.
- Kinetic analysis revealed low micromolar Km values and substrate inhibition patterns.
- NMR confirmed regiospecific phosphorylation at the 3 '-hydroxyl group.
- The deoxystreptamine ring is critical for substrate binding.
Conclusions:
- APH(3 ')-IIIa is a significant contributor to aminoglycoside resistance.
- Detailed mechanistic and structural insights provide a foundation for developing new antibiotic resistance inhibitors.