Evaluating and Predicting Biocatalysts Stereoselectivity using Biomolecular Simulation

Abstract

Enzymes are often stereospecific: they can help to produce one specific stereoisomer through
enzyme-catalysed reactions. Such stereospecificity is important for the application of enzymes
as biocatalysts in industry, especially for drug synthesis, where chiral purity is crucial.
Appropriate enzymes are needed to produce desired compounds and it is not always possible
to find these enzymes. Therefore, enzymes can be engineered to produce the desired
stereoisomer of a compound. To study the origin of enzyme stereoselectivity, two different
aspects of enzyme-catalysed reactions are considered in this thesis. First, the substrate binding
process can determine a binding preference between substrate and enzyme. Depending on the
reaction, a clear preference for a substrate orientation in the active site could make an enzyme
stereoselective. Second, enzyme catalysis is the process where the enzyme lowers the free
energy barrier of the chemical step. If an enzyme does this more effectively for the reaction
forming one stereoisomer, then this can also make an enzyme stereoselective. The first aspect
can be investigated by classical molecular dynamics (MD) simulations of enzyme-substrate
complexes, and (differences in) binding affinities as well as frequency of reaching ‘reactive
poses’ of prochiral substrate binding poses can be calculated. For the second aspect, one can
compare the energy difference for the formation of each stereoisomer. In this thesis, hybrid
semiempirical quantum / classical (QM/MM) MD simulations with umbrella sampling and
QM/MM climbing image-nudged elastic band (CI-NEB) calculations are applied to obtain free
energy profiles and potential energy profiles, respectively. Three different biocatalysts and
their engineered variants are investigated: a reductive aminase (AdRedAm), an aldolase
(SsKDG-aldolase) and a Diels-Alderase (Cyc15). AdRedAm and its variants catalyse a
NADPH-dependent reduction of 2-phenylpyrroline with diastereoselectivities, which is found
to be mostly related to the difference in free energy barrier for the formation of the two
enantiomers. SsKDG-aldolase and three structure-guided variants catalyse the carbon-carbon
bond formation between D-/L- glyceraldehyde and pyruvate. Here, QM/MM CI-NEB
calculations successfully capture the approximate transition states, which indicate that the
observed stereoselectivity is directly related to the calculated difference in energy barrier for
the reaction. Cyc15 catalyses the intramolecular Diels-Alder (DA) reaction of the Omethylated substrate of the Diels-Alderase AbyU, resulting in a different stereoisomer than
that produced by AbyU itself. Here, the binding affinity preference and free energy barrier
difference are identified as potential factors. Overall, the outcome of the final stereoselectivity
of an enzyme-catalysed reaction always involves various subtle effects, which may not always
be captured easily with biomolecular simulations. While studying these three biocatalysts,
biomolecular simulation protocols are developed based on suitable, computationally efficient,
techniques. The protocols aim to understand the sources of the observed enzyme
stereoselectivity and can further be used for predicting and screening novel variants with
desired selectivity efficiently, prior to performing experiments.

Date of Award	4 Feb 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Marc W Van der Kamp (Supervisor), Paul Race (Supervisor) & Christopher Dempsey (Supervisor)