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Author Notes:

Correspondence: Ichiro Matsumuramail; E: imatsum@emory.edu

Authors' Contributions: Conceived and designed the experiments: JRK and IM.

Performed the experiments: JRK.

Analyzed the data: JRK and IM.

Contributed reagents/materials/analysis tools: JRK and IM.

Wrote the paper: JRK and IM.

Acknowledgments: The work described here was inspired in part by an earlier effort by Kristen Marshall Thompson.

We thank Anton Bryksin for the parental aph(3′)-IIIa-pQBAV3c plasmid, and for his advice on protein purification.

We are grateful to Sylvia Yip for her advice pertaining to the enzyme assays.

Disclosures: The authors have declared that no competing interests exist.

Subjects:

Research Funding:

The work described here was inspired in part by an earlier effort by Kristen Marshall Thompson.

We thank Anton Bryksin for the parental aph(3′)-IIIa-pQBAV3c plasmid, and for his advice on protein purification. We are grateful to Sylvia Yip for her advice pertaining to the enzyme assays.

Directed Evolution of Aminoglycoside Phosphotransferase (3′) Type IIIa Variants That Inactivate Amikacin but Impose Significant Fitness Costs

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Journal Title:

PLoS ONE

Volume:

Volume 8, Number 10

Publisher:

, Pages e76687-e76687

Type of Work:

Article | Final Publisher PDF

Abstract:

The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3′) type IIIa (hereafter abbreviated APH(3′)-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3′)-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme) to over 1200 micrograms/mL (clone 4.1). The artificially evolved 4.1 APH(3′)-IIIa variant exhibited 19-fold greater catalytic efficiency (kcat/KM) than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3′)-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density) compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3′)-IIIa in clinical settings.

Copyright information:

© 2013 Kramer, Matsumura.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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