Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

Abstract

Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by beta-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-beta-lactamase (MbetaL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MbetaLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.

Fig. 1.

Active sites of WT BcII and mutant M5. (Upper Left) Side view of a representation of the active site of BcII, in which the protruding loops L3 and L10 are highlighted in orange and yellow, respectively. (Upper Right) Comparison of the active sites of WT BcII and mutant M5. The Zn2 ion in M5 (in green) is more solvent exposed than the Zn2 ion in WT BcII (in gray). The molecular surface of WT BcII is depicted in transparent gray. (Lower) Upper views of the active sites of WT BcII (Left) and M5 (Right) depicting the metal ions, metal ligands, and loops. The long loops flanking the active site (L3 and L10) as well as the loops containing the metal ligands (L7, L9, and L12) are depicted, together with relevant residues. Loops are indicated in diagram tubes. Zn(II) ions are shown as blue spheres. Metal ligands are shown in lines, residues involved in H bonds are shown in sticks, and H bonds are indicated by black dashed lines. The figures were rendered by using PyMol (http://pymol.sourceforge.net/).

Fig. 2.

Catalytic efficiencies of different BcII forms. Relative catalytic efficiencies of G262S BcII, N70S BcII, and N70S/G262S BcII compared with WT BcII against benzylpenicillin (PEN), nitrocefin (NTR), imipenem (IMI), cefotaxime (CTX), and cephalexin (CLX). The relative activity is the ratio of the mutant's k_cat/K_M value to that of WT. y axis is in log scale.

Fig. 3.

Stabilization of a reaction intermediate by mutation G262S. Reaction of BcII WT and G262S with nitrocefin, measured by using a photodiode array detector in a stopped-flow system under single-turnover conditions. All spectra are shown until the reaction completion time. The absorbance changes at 390 (S), 490 (P), and 665 (EI) nm are indicated with arrows. Nitrocefin hydrolysis by WT BcII proceeds by direct conversion of S to P (Left). The G262S mutation stabilizes the anionic intermediate EI (Right), depicted in the reaction scheme.

Fig. 4.

Organismal fitness parallels catalytic efficiency toward cephalexin. Plots of the minimum inhibitory concentrations (MIC) of cephalexin toward E. coli cells expressing the different mutants (Left) compared with the in vitro catalytic efficiencies of the purified enzymes vs. cephalexin (Right).