Validating computer simulations of enantioselective catalysis; reproducing the large steric and entropic contributions in Candida Antarctica lipase B

Abstract

The prospect for computer aided refinement of stereoselective enzymes is further validated by simulating the ester hydrolysis by the wild type and mutants of CalB, focusing on the challenge of dealing with strong steric effects and entropic contributions. This was done using the empirical valence bond (EVB) method in a quantitative screening of the enantioselectivity, considering both kcat and kcat/KM of the R and S stereoisomers. Although the simulations require very extensive sampling for convergence they give encouraging results and major validation, indicating that our approach offers a powerful tool for computer aided design of enantioselective enzymes. This is particularly true in cases with large changes in steric effects where alternative approaches may have difficulties in capturing the interplay between steric clashes with the reacting substrate and protein flexibility.

Understanding Potentials and Restrictions of Solvent‐Free Enzymatic Polycondensation of Itaconic Acid: An Experimental and Computational Analysis

“…Generally speaking, the esterification catalyzed by CaLB follows a ping‐pong bi‐bi mechanism 34. The enzyme stabilizes the negatively charged oxyanion thanks to electrostatic interactions between Thr40 and Gln106 inside the so‐called oxyanion hole.…”

“…Only the tran s CHDM was considered for the docking study since it is the predominant stereoisomer used in the polycondensation mixture. AutoDock version 4.234 was used for performing the docking calculations with Lamarckian genetic algorithm. 100 docking generations, each one composed of 250 docking poses were generated.…”

Computer aided enzyme design and catalytic concepts

“…In particular, we have been successful in direct mutations from R to S [75]. More recently [76] we succeeded to reproduce the effect of large steric effects on the selectivity of CALB [77], a challenge that has not been met by other approaches. Furthermore, the combination of the EVB with our restraint release approach [78] reproduced the entropic contribution to selectivity.…”

Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear β‐Haloalkanes: Open–Solvated versus Occluded–Desolvated Active Sites

“…Enzymatic catalysis is apowerful tool for preparing optically pure chemicals. [1][2][3][4] However,e nzyme engineering is often required to develop biocatalysts that are enantioselective with non-natural substrates.C omputational methods can increase the effectiveness of engineering efforts, [5][6][7][8][9][10] but even when such methods are applied, an understanding of the factors that govern the interactions of the active site with enantiomers is essential for rational engineering.…”