Protein design

Enzyme

Active site

Stereochemistry

Directed evolution

Gene

Mutant

Energy landscape

Catalysis

Computational chemistry

Protein engineering

Chemical physics

Chemistry

Borohydride

Conformational change

Protein structure

Molecular dynamics

Biochemistry

Enzyme catalysis

Transition state

Science

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.

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