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Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

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Aminoglycoside 2''-phosphotransferase type IIIa from Enterococcus.

Adriana Badarau¹, Qicun Shi, Joseph W Chow

¹Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

The Journal of Biological Chemistry

|January 18, 2008

Summary

This summary is machine-generated.

Aminoglycoside phosphotransferases confer antibiotic resistance. This study details a novel enzyme (type IIIa) that uniquely uses guanosine triphosphate, impacting enterococcal infection treatments.

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Antibiotic Dereplication Using the Antibiotic Resistance Platform

Antibiotic Dereplication Using the Antibiotic Resistance Platform

Published on: October 17, 2019

Area of Science:

Biochemistry
Molecular Biology
Microbiology

Background:

Aminoglycoside 2''-phosphotransferases are crucial for high-level aminoglycoside resistance in Gram-positive bacteria.
This resistance poses a significant challenge for treating severe enterococcal infections.

Purpose of the Study:

To clone, purify, and characterize the mechanism of an aminoglycoside 2''-phosphotransferase (formerly type Ic).
To investigate its substrate specificity and catalytic mechanism.

Main Methods:

Enzyme cloning and purification.
Enzymatic assays to determine kinetic parameters (kcat, Km).
Inhibition studies (product and dead-end) and solvent viscosity effects.
Computational modeling.

Main Results:

The enzyme unexpectedly prefers guanosine triphosphate over adenosine triphosphate as a phosphate donor.
It phosphorylates specific 4,6-disubstituted aminoglycosides at the 2''-hydroxyl position with nanomolar affinity.
The enzyme mechanism was determined to be random sequential Bi Bi, with guanosine triphosphate release being rate-limiting at pH 7.5.
Renamed the enzyme aminoglycoside 2''-phosphotransferase type IIIa based on its unique profile.

Conclusions:

Aminoglycoside 2''-phosphotransferase type IIIa possesses a unique biochemical profile, particularly its guanosine triphosphate preference.
Understanding this mechanism provides insights into antibiotic resistance and potential therapeutic strategies against enterococcal infections.