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Light-Emitting Dehalogenases Research Article Cite This: ACS Catal. 2019, 9, 4810−4823 pubs.acs.org/acscatalysis Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts Radka Chaloupkova,† Veronika Liskova,† Martin Toul,† Klara Markova,† Eva Sebestova,† Lenka Hernychova,‡ Martin Marek,† Gaspar P. Pinto,†,∥ Daniel Pluskal,† Jitka Waterman,§ Zbynek Prokop,†,∥ and Jiri Damborsky*,†,∥ † Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic ‡ Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic § Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom ∥ International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic *S Supporting Information ABSTRACT: To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Its crystal structure solved to 1.39 Å resolution revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descend- ants and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen−deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts. Downloaded via MASARYK UNIV on July 30, 2020 at 06:04:28 (UTC). KEYWORDS: catalytic promiscuity, ancestral reconstruction, haloalkane dehalogenase, monooxygenase, luciferase, emergence of biological function See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ■ INTRODUCTION refer to different chemistries and different classes of substrates or even by the first digit, indicating a completely different Under selective pressures, enzymes have generally developed 6 high catalytic efficiency and specificity. Hence, they are reaction category. The emergence of enzymatic functions has fi often been associated with the initial occurrence of functionally classi ed in terms of the main substrate 8 conversions they catalyze in vivo (their “native reactions”). promiscuity, and understanding enzyme promiscuity is However, many have “catalytic promiscuity”, i.e., the ability to becoming increasingly important to improve our general catalyze reactions involving non-natural substrates or addi- knowledge of enzyme evolution and ability to engineer − tional reactions.1 4 The degree of an enzyme’s promiscuity is enzymes with unique properties. Ancestral enzymes putatively had catalytic promiscuity, generally assessed by examining the types of bonds formed or “ ” fi broken during its catalytic activities and differences in being generalists exhibiting broad substrate speci city and, ffi 6,8−11 mechanisms between native and “promiscuous” reactions.5 A at the same time, low catalytic e ciency, but through ’ mutation, duplication, and horizontal transfer, gene families systematic, hierarchical approach is to classify enzymes fi fi ffi promiscuity in terms of differences in Enzyme Commission diversi ed and variants with more speci candecient (EC) numbers of the reactions they can catalyze.6,7 Catalytic promiscuity generally corresponds to the cases in which the EC Received: March 11, 2019 numbers of the various substrates and reactions catalyzed by Revised: April 14, 2019 the same enzyme differ in the second, or the third, digits that Published: April 15, 2019 © 2019 American Chemical Society 4810 DOI: 10.1021/acscatal.9b01031 ACS Catal. 2019, 9, 4810−4823 ACS Catalysis Research Article Figure 1. Distinct reaction mechanisms of closely evolutionary related haloalkane dehalogenases (HLDs, EC 3.8.1.5) and a Renilla luciferase (RLuc, EC 1.13.12.5). (A) HLDs catalyze hydrolytic dehalogenation of halogenated alkanes to a primary alcohol, a halide ion (X−), and a proton. The two-step mechanism is initiated by a nucleophilic attack of aspartic acid on the carbon atom of the substrate resulting in a covalent alkyl enzyme intermediate. This complex is subsequently hydrolyzed by a water molecule activated by catalytic base.24 (B) RLuc catalyzes monooxygenation of coelenterazine in the presence of oxygen, yielding carbon dioxide, blue light, and coelenteramide. The catalytic mechanism of RLuc is poorly understood but is expected to proceed via a dioxetanone intermediate.25,26 − − catalytic activities evolved.6,11 13 Although the ancestral core domain and specificity-determining cap domain.33 35 The enzymes no longer exist, their sequences can be inferred by core domain includes a conserved catalytic pentad composed − ancestral sequence reconstruction.14 16 Reconstructed ances- of a nucleophile, a base, a catalytic acid, and two halide- tral enzymes often exhibit substrate promiscuity. For example, stabilizing residues.27,30 However, the reaction mechanism and resurrected ancestors of α-glucosidases have exhibited hydro- substrate specificity of HLDs and RLuc are strikingly different lytic activity toward broader ranges of maltose-like and (Figure 1). HLDs have a two-step reaction mechanism, isomaltose-like sugars than their modern descendants.12 involving activated water-mediated hydrolysis of a covalently − Similarly, reconstructed ancestors of β-lactamases,17 hydrox- bound alkyl enzyme intermediate.24,36 40 In contrast, the ymethyl pyrimidine kinases,18 carboxyl methyltransferases,19 incompletely understood catalytic mechanism of RLuc and 2-ketoacid oxidoreductases20,21 have all exhibited higher putatively involves the decomposition of a dioxetanone substrate promiscuity than their respective descendants. intermediate that provides energy enabling generation of a However, very few examples of resurrected ancestral enzymes product molecule in an electronically excited state.26,25 exhibiting catalytic promiscuity (i.e., the ability to catalyze Hydrolytic conversion of halogenated alkanes releases an different kinds of reactions, rather than the same kind of alcohol, while monooxygenation of coelenterazine results in reaction with similar substrates) have been reported.22,23 emission of light. A common ancestor of HLDs and RLuc is an Moreover, although many reconstructed ancestral hydroxyni- exciting and challenging target for reconstruction, as it could trile lyases and esterases at four nodes of a published profoundly illuminate elusive aspects of these strongly differing phylogenetic tree can catalyze both an ester hydrolysis and a reaction mechanisms. Here, we combined several methods cyanohydrin cleavage, the extant enzymes also have some including ancestral sequence reconstruction, X-ray crystallog- (albeit weaker) promiscuous activity.22 raphy, steady-state and presteady-state enzyme kinetics, matrix- In the study reported here, we reconstructed a common assisted laser desorption ionization-time-of-flight mass spec- ancestor of haloalkane dehalogenases27 and Renilla lucifer- trometry (MALDI-TOF MS), site-directed mutagenesis, ase,28,29 which are catalytically specific enzymes although they molecular dynamics (MD), and hydrogen−deuterium ex- belong to the same HLD-II enzyme subfamily.30 Haloalkane change mass spectrometry (HDX-MS) to get structural insight dehalogenases (HLDs, EC 3.8.1.5) catalyze hydrolytic into the understanding why evolutionary related modern-day dehalogenation of a broad range of halogenated aliphatic enzymes exhibit distinct catalytic functions (Figure S1). compounds into the corresponding alcohols, a halide anion, and a proton.27,31 Luciferase from Renilla reniformis (RLuc, EC ■ MATERIALS AND METHODS 1.13.12.5)28,29 catalyzes (with no need for any cofactor) Ancestral Sequence Reconstruction. The ancestral monooxygenation of a coelenterazine, yielding a coelenter- sequence was reconstructed as previously described.41 Briefly, amide and a carbon dioxide, with emission of blue light.26,29 a nonredundant set of HLD-II protein sequences was collected RLuc is the only characterized monooxygenase, which is by PSI-BLAST v2.2.22+42 searches against the NCBI nr clustered in the HLD enzyme family together with recently database (version 21-9-2012)43 and then clustered with identified putative luciferase from Amphiura filiformis.32 CLANS44 and CD-HIT v4.5.4.45 The data set was divided Activity of this putative luciferase with coelenterazine has into two subgroups based on their evolutionary relatedness. been detected with the crude extract from arms of the For each HLD-II subgroup, a separate multiple sequence animal,32 while production of light by the purified protein alignment (MSA) was constructed by MUSCLE v3.5146 and needs to be verified. Structurally, both
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