Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels

Javier Iglesias-Fernández,Sílvia Osuna,Miquel Estévez-Gay

doi:10.3390/catal10121403

Javier Iglesias-Fernández, Sílvia Osuna + Show 1 more

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https://doi.org/10.3390/catal10121403

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Abstract

Halohydrin dehalogenases (HHDH) are industrially relevant biocatalysts exhibiting a promiscuous epoxide-ring opening reactivity in the presence of small nucleophiles, thus giving access to novel carbon–carbon, carbon–oxygen, carbon–nitrogen, and carbon–sulfur bonds. Recently, the repertoire of HHDH has been expanded, providing access to some novel HHDH subclasses exhibiting a broader epoxide substrate scope. In this work, we develop a computational approach based on the application of linear and non-linear dimensionality reduction techniques to long time-scale Molecular Dynamics (MD) simulations to study the HHDH conformational landscapes. We couple the analysis of the conformational landscapes to CAVER calculations to assess their impact on the active site tunnels and potential ability towards bulky epoxide ring opening reaction. Our study indicates that the analyzed HHDHs subclasses share a common breathing motion of the halide binding pocket, but present large deviations in the loops adjacent to the active site pocket and N-terminal regions. Such conformational differences affect the available tunnels for epoxide binding to the active site. The superior activity of the HHDH G subclass towards bulkier substrates is explained by the additional structural elements delimiting the active site region, its rich conformational heterogeneity, and the substantially wider and frequently observed active site tunnels. This study therefore provides key information for HHDH promiscuity and engineering.

Highlights

Enzymes are highly efficient in accelerating the chemical reactions under biologically controlled conditions, and can provide synthetically useful building blocks with high selectivity and specificity.The ability of enzymes of accelerating additional side reactions, i.e., they present catalytic promiscuity, is thought to play a key role in the evolution of enzymes towards new functions [1,2]
Our study indicates that the analyzed HHDHs subclasses share a common breathing motion of the halide binding pocket, but present large deviations in the loops adjacent to the active site pocket and N-terminal regions
Our study starts with the evaluation of the conformational landscapes of the different Halohydrin dehalogenase (HHDH) variants from the subclasses A2, B, C, D2, and G, followed by a rationalization of how dynamics affect their promiscuity towards epoxide ring opening [22,23,24]

Summary

Introduction

Enzymes are highly efficient in accelerating the chemical reactions under biologically controlled conditions, and can provide synthetically useful building blocks with high selectivity and specificity.The ability of enzymes of accelerating additional side reactions, i.e., they present catalytic promiscuity, is thought to play a key role in the evolution of enzymes towards new functions [1,2]. The appearance of novel enzyme functionalities through evolution has been attributed to the fine-tuning of the conformational ensemble present in solution, whose relative stabilities can be tuned by mutations [3,4,5,6,7] Many of these pre-existing conformations can play a key role in recognizing and binding the substrate and/or releasing the product, in conferring the enzyme the catalytic promiscuity, and in some cases in regulating the operating allosteric communication. These additional conformations of the enzyme can present deviations in the available tunnels for accessing the active site, playing a role in the enzyme catalytic activity. The engineering of some flexible loops gating substrate access to Catalysts 2020, 10, 1403; doi:10.3390/catal10121403 www.mdpi.com/journal/catalysts

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