Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels

catalysts Article Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels 1 1, , 1,2, Miquel Estévez-Gay , Javier Iglesias-Fernández * y and Sílvia Osuna * 1 CompBioLab Group, Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, c/Maria Aurèlia Capmany 69, 17003 Girona, Catalonia, Spain; [email protected] 2 ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Catalonia, Spain * Correspondence: [email protected] (J.I.-F.); [email protected] (S.O.) Present address: Nostrum Biodiscovery, Carrer de Baldiri Reixac, 10–12, 08028 Barcelona, Catalonia, Spain. y Received: 12 November 2020; Accepted: 28 November 2020; Published: 1 December 2020 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. Keywords: Halohydrin dehalogenases; conformational dynamics; active site tunnels; molecular dynamics simulations 1. 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 [3–7]. 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, thus playing a role in the enzyme catalytic activity. Indeed, the engineering of some flexible loops gating substrate access to Catalysts 2020, 10, 1403; doi:10.3390/catal10121403 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 15 additional conformations of the enzyme can present deviations in the available tunnels for accessing the active site, thus playing a role in the enzyme catalytic activity. Indeed, the engineering of some flexibleCatalysts loops2020, 10 gating, 1403 substrate access to the active site and contributing to product release were2 of 14 shown to be key for boosting the catalytic activity of some enzymes [8,9]. The enzyme conformationalthe active site landscape and contributing therefore to plays product a crucial release role were in shownits function, to be promiscuity, key for boosting regulation, the catalytic and evolution.activity of some enzymes [8,9]. The enzyme conformational landscape therefore plays a crucial role in its function,Halohydrin promiscuity, dehalogenases regulation, (HHDHs) and evolution.perform a cofactor independent dehalogenation reaction for degradingHalohydrin halogenated dehalogenases compounds. (HHDHs) They perform are a cofactorhighly valuable independent biocatalysts dehalogenation as they reaction exhibit for promiscuousdegrading halogenated epoxide ring compounds.-opening catalytic They are activity highly valuable in the presence biocatalysts of assmall they nucleophiles, exhibit promiscuous thus givingepoxide access ring-opening to novel catalyticcarbon–carbon, activity incarbon the presence–oxygen, of carbon small nucleophiles,–nitrogen, and thus carbon giving–sulfur access tobonds novel [10,11]carbon–carbon,. Some biocatalytic carbon–oxygen, examples carbon–nitrogen, of HHDH-catalyzed and carbon–sulfur reactions bondsinclude [10 their,11]. Someapplication biocatalytic for obtainingexamples ofstatin HHDH-catalyzed side chains reactionsprecursors include more their efficiently, application forenantiopure obtaining statinepihalohydrins, side chains oxazolidinones,precursors more tertiary efficiently, and enantiopure beta-substitu epihalohydrins,ted alcohols oxazolidinones,[12–18]. As shown tertiary by andthe beta-substitutedsolved X-ray structures,alcohols[ 12the– 18active]. As site shown of HHDH by the solvedis composed X-ray structures,by a binding the site active for sitethe ofepoxide HHDH and is composed a spacious by halidea binding binding site pocket for the that epoxide can accommodate and a spacious linear halide monovalent binding anions pocket as that nucleophiles can accommodate (see Figure linear 1) [19,20]monovalent. HHDH anionss feature as nucleophiles a conserved (see catalytic Figure 1triad)[ 19 ,20composed]. HHDHs by feature Ser-Tyr a conserved-Arg, which catalytic catalyzes triad epoxidecomposed formation by Ser-Tyr-Arg, and subsequent which catalyzes halide release epoxide [19,20] formation. The andpromiscuous subsequent epoxide halide ring release-opening [19,20 ]. reaction usually occurs at the less-hindered carbon via SN2 mechanism [20], and the range of The promiscuous epoxide ring-opening reaction usually occurs at the less-hindered carbon via SN2 epoxidesmechanism accepted [20], by and HHDHs the range is usually of epoxides limited accepted to terminal by HHDHs epoxides is [21] usually, although limited some to terminalrecent examplesepoxides of [21 HHDH], although accepting some recent sterically examples more of demanding HHDH accepting epoxides sterically have morebeen demandingreported [22] epoxides (see Schemehave been 1). reportedIn a recent [22 ]paper (see Scheme by the1 ).Schallmey In a recent lab, paper novel by theHHDHs Schallmey were lab,identified novel HHDHsfollowing were a databaseidentified mining following approach, a database with miningsix phylogenetic approach, subtypes with six phylogenetic of HHDH ranging subtypes from of HHDHA through ranging G characterizedfrom A through [23] G. Particularly characterized useful [23]. is Particularly HheG, as usefulit represents is HheG, the as first it represents example of the HHDH first example able to of acceptHHDH with able synthetically to accept with useful synthetically activities usefulbulky cyclic activities epoxides bulky as cyclic substrates epoxides [22,23] as substrates. Interestingly [22,23, ]. HheGInterestingly, has also HheG been has recently also been found recently to foundexhibit to exhibithigh activity high activity towards towards sterically sterically demanding demanding didi-substituted-substituted epoxides,epoxides, whereas whereas the A-Fthe subtypes A-F presentsubtypes activity present only towardsactivity methyl-disubstituted only towards methylepoxide-disubstit substratesuted [24 epoxide] (see Scheme substrates1). Unfortunately, [24] (see structureScheme(i.e., 1). conformationalUnfortunately, dynamics)–activity structure (i.e., conformationalrelationships are dynamics) not available–activity for thisrelationships family of are enzymes. not available for this family of enzymes. Figure 1. Halohydrin dehalogenase (HHDH) distinct structural elements and zoom of the active site Figure 1. Halohydrin dehalogenase (HHDH) distinct structural elements and zoom of the active site and halide binding pockets based on the HheC structure. Active site residues are highlighted in and halide binding pockets based on the HheC structure. Active site residues are highlighted in wheat color, halide-binding site in teal, N-terminal loop in light green, C-terminal loop in purple and wheat color, halide-binding site in teal, N-terminal loop in light green, C-terminal loop in purple and N-terminal 6–7 helices in salmon. In the active site zoom, potential residues blocking the accessible active site tunnels are depicted using the same color scheme. Catalysts 2020, 10, x FOR PEER REVIEW 3 of 14 Catalysts 2020, 10, 1403 3 of 14 Scheme 1. (A). Reaction scheme of the two-step HHDH catalyzed enzymatic reaction: (1, left) Scheme 1. (A). Reaction scheme of the two-step HHDH catalyzed enzymatic reaction: (1, left) dehalogenation for epoxide formation, followed by the promiscuous (2, right) enantioselective dehalogenation for epoxide formation, followed

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