title: 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. 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.
authors.affiliation: Emory University School of Medicine, Department of Biochemistry, Rollins Research Center, Atlanta, Georgia, United States of America.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Kramer
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsumura
mesh_terms: D000583:Amikacin / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D056084:Genetic Fitness; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009154:Mutation / Q000187:drug effects*; D017434:Protein Structure, Tertiary
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D000583:Amikacin; D019868:Kanamycin Kinase