Selection of allosteric beta-lactamase mutants featuring an activity regulation by transition metal ions

Abstract

Libraries of phage-displayed beta-lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild-type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double-selection protocol was applied: The phage-displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal-specific manner. A remarkable mutant exhibiting differential allosteric regulation depending on the metal was found. Its activity was activated by nickel ion binding, inhibited by cupric ion binding, and nearly unaffected by zinc ions. These observations point to an interesting potential for up- or down-regulation of activity within a monomeric enzyme by binding to an "allosteric site" relatively remote from the active site.

Figure 1.

Ratios between the numbers of phages eluted versus the numbers of phages loaded on metal ion–NTA-coated magnetic beads. (A) Increase of the elution ratios with selection rounds on NTA–Ni²⁺ beads. (B) Elution ratios for libraries preselected on NTA–Cu, NTA–Ni, or NTA–Zn and reselected on the same or a different support.

Scheme 1.

Figure 2.

Effect of Ni²⁺ (♦) or Zn²⁺ (▴) ion concentrations on activities (k _cat) of mutants A5, A7, and A13 in which four histidine residues are distributed differently in the three engineered loops. The lines were calculated using Equation 2 and the parameters given in Table 2.

Scheme 2.

Figure 3.

Model of mutant A5. The engineered loops are shown in red with the histidine residue side chains displayed as balls and sticks. The segment of loop L3 containing the original residues is shown in magenta. The nucleophilic serine of the active site is displayed in space filling. Two orthogonal views are shown.