Directed evolution of aminoglycoside phosphotransferase (3') type IIIa variants that inactivate amikacin but impose significant fitness costs.

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 (k cat/K M) than did the wild-type enzyme in reactions with amikacin. E. c

SUBMITTER: Kramer JR 

PROVIDER: S-EPMC3800079 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature