Role of Conformational Dynamics in the Evolution of Retro-Aldolase Activity

Abstract

Enzymes exist as ensembles of conformations that are important for function. Tuning these populations of conformational states through mutation enables evolution toward additional activities. Here we computationally evaluate the population shifts induced by distal and active site mutations in a family of computationally designed and experimentally optimized retro-aldolases. The conformational landscape of these enzymes was significantly altered during evolution, as pre-existing catalytically active conformational substates became major states in the most evolved variants. We further demonstrate that key residues responsible for these substate conversions can be predicted computationally. Significantly, the identified residues coincide with those positions mutated in the laboratory evolution experiments. This study establishes that distal mutations that affect enzyme catalytic activity can be predicted computationally and thus provides the enzyme (re)design field with a rational strategy to determine promising sites for enhancing activity through mutation.

Conformational dynamics and enzyme evolution

“…Interface 15: 20180330 the 23 mutations introduced during the directed evolution were located at positions that were distant from the active site, indicating that their impact is rather to influence the overall evolution of conformational dynamics of the enzyme. In particular, the conformational dynamics of flexible loops on the catalytic face of the TIM-barrel fold were affected by mutations, as shown by MD simulations [26]. Over the evolutionary trajectory of RA95, these loops were observed to become substantially more rigid, while the overall enzymes themselves gained increased thermostability [26,83].…”

“…The importance of conformational dynamics for the evolution of enzyme activity was also showcased using MD simulations of a range of engineered retro-aldolases (RAs) [26]. Several RAs were designed de novo to break the C-C bond in unnatural substrate 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone (methodol) [79,80].…”

“…In the case of RA95, further directed evolution of the original design increased the activity by another six orders of magnitude over 19 rounds of directed evolution, and with the mutations at 23 different amino acid positions (figure 5) [82,83]. Osuna and co-workers studied the evolutionary trajectory of RA95, where many new mutations were located far away from the active site, yet they still had positive impact on the catalytic turnover [26]. Introducing the first six mutations into the scaffold (i.e.…”

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates

“…Computational methods have shown to be particularly useful in this regard . We have recently found that molecular dynamics (MD) simulations coupled to correlation‐based analysis can provide important insights on the alternative conformations that the enzyme can adopt in solution, but most importantly can predict which amino acid positions are key for favoring a desired population shift to enhance a promiscuous activity …”

An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols

“…Mutation of these residues might affect the position of the loop and hence substrate access to the active site. Any further increase in activity will probably require more mutations that are distal to the active site and could be predicted computationally …”