Computational Prediction of ω-Transaminase Specificity by a Combination of Docking and Molecular Dynamics Simulations

Abstract

ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from Vibrio fluvialis with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined ee% values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.

Molecular Evolution of an Aminotransferase Based on Substrate–Enzyme Binding Energy Analysis for Efficient Valienamine Synthesis

“…Here, we chose the amino acceptor−PMP external aldimine intermediate to explore different catalytic activities of RffA_Kpn toward TDP-Glc4O and valienone because the amino acceptor−PMP external aldimine is a stable and defined intermediate that is often co-crystallized with ATs 37,38 and preferred for computational mechanism exploration and activity engineering of ATs. 25,39,40 The binding conformation and the interaction of the external aldimine intermediates TDP-Glc4O−PMP and valienone−PMP in RffA_Kpn were compared and analyzed after 100 ns molecular dynamics (MD) simulations. 10 residues from the homodimer (A: C55, T56, T84, S176, K181, H320, Y321, and R352; and B: R213 and Y224) were predicted to be involved in the binding of TDP-Glc4O−PMP by forming 16 hydrogen bonds, including two with Glc4O, seven with PMP, and seven with the TDP moiety (Figures 2A and S5).…”

“…A nucleophilic attack on the external aldimine by K181 requires the reacting atoms to be positioned at a short distance. 24,39 Higher activity toward valienone would likely occur when valienone−PMP is bound in the "Glc4O− PMP region" of the binding pocket with a similar conformation to the Glc4O−PMP portion of the native reaction. Thus, efforts should be devoted to adapting the catalytic conformation of valienone−PMP to achieve improved catalytic activity for valienamine synthesis.…”

“…Next, the reaction is reversed to form an external aldimine between PMP and the amino acceptor (amino acceptor–PMP), after which the amine product is released, and E:PLP is regenerated (Scheme S1). Here, we chose the amino acceptor–PMP external aldimine intermediate to explore different catalytic activities of RffA_Kpn toward TDP-Glc4O and valienone because the amino acceptor–PMP external aldimine is a stable and defined intermediate that is often co-crystallized with ATs , and preferred for computational mechanism exploration and activity engineering of ATs. ,, …”

“…The apparent binding deviation between valienone–PMP and TDP-Glc4O–PMP in RffA_Kpn (Figures D and S6B) was caused by valienone–PMP binding in a non-ideal position during catalysis in the oversized binding pocket of the enzyme, which supports the low transaminase activity toward valienone observed experimentally. A nucleophilic attack on the external aldimine by K181 requires the reacting atoms to be positioned at a short distance. , Higher activity toward valienone would likely occur when valienone–PMP is bound in the “Glc4O–PMP region” of the binding pocket with a similar conformation to the Glc4O–PMP portion of the native reaction. Thus, efforts should be devoted to adapting the catalytic conformation of valienone–PMP to achieve improved catalytic activity for valienamine synthesis.…”

Activity Levels of Amine Transaminases Correlate with Active Site Hydrophobicity

“…The different sizes of the binding pockets explain the stereopreference of ATAs, in case that the substrate bears two differently sized substituents, since one orientation of the substrate fits better to the shape of the pockets and thus is energetically favorable. Based on this principle, molecular docking algorithms combined with molecular modeling can be used to predict the enantioselectivity and substrate specificity of transaminases with known structures [22].…”