Ground-State Destabilization by Active-Site Hydrophobicity Controls the Selectivity of a Cofactor-Free Decarboxylase.

Michal Biler,Robert Kourist,Shina Caroline Lynn Kamerlin,Anna K Schweiger,Rory M Crean

doi:10.1021/jacs.0c10701

Michal Biler, Robert Kourist + Show 3 more

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https://doi.org/10.1021/jacs.0c10701

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Abstract

Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.

Highlights

Enzymatic catalysis of the formation and breaking of C−C bonds is currently receiving increasing attention.[1]
We note that as the grid inhomogeneous solvation theory (GIST) analysis was performed on the unliganded states of each enzyme, and the optimal positions of both carboxyl groups are essentially identical across the different substrates for the same binding pose, we focused our GIST analysis on only compound 1b
We considered the solvent-accessibility of the active site in our simulations, taking into account that one of the two carboxylate groups is stabilized by a dioxyanion hole while the other carboxylate group is located in a hydrophobic pocket

Summary

Introduction

Enzymatic catalysis of the formation and breaking of C−C bonds is currently receiving increasing attention.[1]. With its highly unusual mechanism, orotidine-5′-phosphate decarboxylase has emerged as a model to study enzymes using ground-state destabilization as a catalytic principle.[12] Among several discussed mechanisms, one uses a so-called “Circe”-effect, in which binding of the phosphate group accommodates the substrate in a binding mode where unfavorable interactions lead to cleavage of a carboxylate group of the substrate In this vein, the mechanism of phenolic acid decarboxylase (PAD) has been suggested to proceed via a quinone methide intermediate formed by protonation of the substrate double bond.[3] This explicitly requires hydrogen bonding of the p-hydroxy group of the substrate with two tyrosine residues. PAD decarboxylates differently substituted cinnamic acid derivatives, but all substrates must bear a p-hydroxy group.[1,13]

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