How directed evolution reshapes the energy landscape in an enzyme to boost catalysis

Abstract

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. The development of protein catalysts for many chemical transformations could be facilitated by explicitly sampling conformational substates during design and specifically stabilizing productive substates over all unproductive conformations.

Fig. 1.. Inactivation of Kemp eliminase variants is due to sampling of an alternative, folded conformation.

(A) The Kemp elimination reaction (8) with the structure of the transition-state analog shown on the right. (B) Temperature- and pH-dependent NMR experiments for free HG3.17 display peak duplication for many residues (Fig. S2A) as exemplified here for Gly263. The cross peak of the minor, inactive (I) species increases with temperature and/or pH, indicative of a slow interconversion process between two folded conformations. (C) Directed evolution greatly increased catalytic efficiency (k_cat/K_M) from HG3 to HG3.17 (3), but for evolved enzymes a clear temperature-dependent inactivation is observed. (D) Protein stability measurements using thermal-shift assays indicate that inactivation above ~25 °C is not due to global unfolding, but the smaller transition at lower temperatures suggests the presence of another state.

Fig. 2.. Characterization of the inactive/active interconversion of Kemp eliminase variants.

(A) Active and inactive conformations are observed for all HG3 variants as exemplified by the NMR cross peaks of Gly229 at pH 7. At 25 °C the inactive population is small for HG3.17, but sizeable for HG3 and HG3.7, and the inactive species increases with temperature. (B-D) Detection of interconversion kinetics at 25 °C by real-time NMR using a pH-jump from proteins equilibrated at pH 10.0 to 7.0. (B) pH-jump experiment for HG3.7 followed by 2D HSQC spectra confirm that the interconversion from the inactive (at high pH) to active (at lower pH) conformation indeed occurs, but the quality of the data is insufficient to extract reliable rate constants. (C, D) The measurements were repeated using 1D proton NMR experiments and time-dependent changes of selected peak areas are shown (C). Observed rate constants (k_obs) in (C), combined with the populations from NMR (A), yielded the activation rate constant (k_ina→act; D).

Fig. 3.. X-ray data reveal extensive structural changes between the active and inactive conformations of the Kemp eliminases.

(A-E) Crystal structures in the absence of TSA show two conformations for residues near the active site of HG3 (A) and HG3.7 (B), but not of HG3.17 (C). The active state (light colors) makes favorable interactions with the modeled TSA (transparent gray; A-C), whereas the inactive state (dark colors) is a binding-incompetent conformation as the carbonyl-group of Leu236 would clash with the TSA (D). (E) The active conformation of free HG3.7 is nearly superimposable with its TSA-bound form. (F) The inactive backbone conformation is the only one observed in the xylanase scaffold (red, PDB 1gor (14)). (G) X-ray structure of inactive conformation of HG3.17, obtained by calcium (green) binding at a surface-exposed site. Residues with NMR peak duplication (Fig. 1B) are shown in blue, unassigned residues in grey, and prolines in black. (H) Superposition of the active (yellow) and inactive, calcium-bound (orange) conformation of HG3.17 shows the propagation of backbone changes from the calcium-binding site extending to the active site with modeled TSA. (I) The mFo-DFc-polder map (green mesh, contoured at 3σ) for crystallographic data recorded at 70 °C for free HG3.17 can only be explained by modeling both the active (yellow) and inactive (orange) conformations (see also Fig. S7H).

Fig. 4.. Transition-state analogue binding as a proxy for probing the chemical activation barrier over evolution.

(A) Mechanism and microscopic equilibrium constants (reported as dissociation constants) for TSA binding to the HG3 variants. (B) Kinetic parameters obtained by numerically fitting the progress curves for substrate conversion at 25 °C to an extended Michaelis-Menten model (Fig. S14A). (C) The increase in (K_S/k_cat)·k_uncat through the evolutionary rounds correlates remarkably well with the change in K₂, as expected from transition-state theory. (D) Ensemble refinements (see also Fig. S16-S17) of cryogenic X-ray structures of HG3 variants bound to TSA reveal extensive conformational sampling for HG3 and HG3 K50Q, whereas in evolved enzymes the side chain orientations become more ordered leading to optimal positioning of the catalytic base Asp127 and the oxyanion stabilizer Gln50 (see also Fig. S15-S16). The apparent order for residues Lys50, Trp87, Ser89, and Gln90 in HG3 is explained by crystal contacts in that region (Fig. S16D) that are specific to HG3. HG3 K50Q is thus better suited for comparison of the ensembles as it forms similar crystal contacts as HG3.7 and HG3.17. (E) k_cat values for all Kemp eliminase variants (Fig. S18) highlight the major boost in k_cat by the K50Q/M84C substitutions.