Table 1.
Primers used in this study.
Table 2.
Aph(3′)-IIIa sequences and minimum inhibitory concentrations (round 1).
Table 3.
Aph(3′)-IIIa sequences and minimum inhibitory concentrations (rounds 2–4).
Figure 1.
Model of artificially evolved aminoglycoside phosphotransferase (3′)-IIIa, based upon crystal structure (1L8T) of the wild-type enzyme [23] rendered in PyMOL.
The putative beneficial mutations are colored orange, the catalytic D190 residue is green), adenosine diphosphate is yellow and magnesium is pink. Kanamycin is blue, while the “extra” moiety that differentiates amikacin from kanamycin is red.
Figure 2.
Michaelis-Menten plots of the wild-type and 4.1 APH(3′)-IIIa variants.
The plots show the dependence of initial velocity upon substrate concentration for the following reactions: a) wild-type APH(3′)-IIIa with kanamycin, b) artificially evolved 4.1 variant with kanamycin, c) wild-type APH(3′)-IIIa with amikacin, and d) 4.1 variant with amikacin. Substrate concentration is in units of micromolar and reaction velocity is in moles of substrate/moles of enzyme/second. Each series of reactions was conducted in triplicate. The average initial velocity values were fit to the Michaelis-Menten equation (a, c) or a simple substrate inhibition model (d) as described in the Methods; the derived kinetic parameters are presented in Table 4.
Table 4.
Kinetic Parameters of the wild-type and evolved 4.1 APH(3′)-IIIa.
Table 5.
Fitness of transformed isogenic Escherichia coli.
Table 6.
Stability of ancestral and evolved aph(3′)-IIIa-pQBAV3c plasmids.