Periodic Reporting for period 1 - Ancestral (Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity) Reporting period: 2018-06-01 to 2020-05-31 Summary of the context and overall objectives of the project Haloalkane dehalogenases (HLDs), which catalyse the cleavage of the carbon-halogen bond of organohalogen compounds, are recognized as key tools in many industrial and biotechnological processes. Interestingly, HLD enzymes display remarkable sequence and structural similarity with luciferase from the marine invertebrate Renilla reniformis (RLuc), which reflects their common evolutionary history. Unlike HLDs, which belong to the family of α/β hydrolases (EC 3.8.1.5) the RLuc luciferase is cofactor-independent monooxygenase (EC 1.13.12.5) that oxidoreductively converts substrate – coelenterazine − into coelenteramide and carbon dioxide, followed by an emission of blue light (470 nm). For this bioluminescent effect, the RLuc luciferase is commonly used as a reporter enzyme in cell biological research and bioimaging technologies.Rational bioengineering attempts to create new RLuc variants with fine-tuned bioluminescent properties are however hampered by the fact that its catalytic reaction mechanism remains poorly understood. This is predominantly due to the lack of atomic-level structural data on catalytically-competent RLuc-substrate and RLuc-product complexes. Poor crystallibility and long-term (>3 months) crystallization process of RLuc appeared to be major drawbacks in the acquisition of structural data that should provide the molecular dissection of RLuc catalytic reaction mechanism.To overcome these limitations, ancestral sequence reconstruction (ASR) represents a powerful approach, in which a hypothetical ancestral sequence of a given present-day enzymes is predicted and reconstructed in a laboratory. The reconstituted ancestral enzymes have been shown to be valuable tools in our understanding of the evolution of biocatalytic mechanisms. Moreover, the enzymes created by ASR often exhibit enhanced thermal stability and promiscuous enzymatic properties, which can be useful in various industrial settings. This project aimed to employ in-lab reconstructed dual-function (dehalogenase/luciferase) ancestral enzyme (ancHLD-RLuc) to decipher molecular evolution steps leading to the functional divergence of modern-day HLD and RLuc enzymes, and to explore how this knowledge could be exploited biotechnologically. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far One of the major intentions of this project was to determine atomic-level structures of the dual-function ancestral enzyme ancHLD-RLuc complexed with bound substrate molecules, with emphasis on coelenterazine-powered monooxygenation (luciferase) catalysis. We succeeded to reconstitute stable complexes of recombinantly-produced and affinity-purified ancHLD-RLuc enzyme complexed with substrate-like and product molecules. We showed that a non-oxidizable coelenterazine analogue binds to the ancHLD-RLuc enzyme with low nanomolar affinity. Unlike the native coelenterazine, the derivative cannot be oxidized, and therefore it represents a perfect chemical tool to study enzyme-substrate complexes. Our crystallographic findings provided unprecedented molecular views of the Renilla-type luciferases complexed with a non-oxidizable coelenterazine analogue, coelenteramide and coelenteramine, revealing as-yet-unseen molecular details of bioluminescent reaction. Specifically, our structures highlight: (i) amino acid residues involved in chemical steps, (ii) residues that deprotonate/reprotonate the coelenteramide to yield blue light (480 nm), and (iii) coordinated motions of enzyme loops carrying aromatic residues that are important for the bulky ligand binding-unbinding turnover. Based on our unique structural data we were able to propose the catalytic mechanism of conversion of coelenterazine into coelenteramide, accompanied with emission of blue light, at alpha/beta-hydrolase fold. In parallel, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) approach to gain molecular insights into ancHLD-RLuc protein dynamics, and identification of conformationally rich regions that could be important for the catalytic reaction. Our results demonstrated that AncHLD-RLuc exhibited a lower level of deuteration for most of the peptides over its amino acid sequence compared to both RLuc and LinB in the shorter reaction times, implying the more compact structure of the enzyme at the beginning of deuteration reaction. The important differences were identified in HD exchange kinetic profiles of the peptides corresponding to the structural elements of the cap domains. Specifically, RLuc exhibited the highest HD exchange kinetics in almost all structural elements forming the cap domain. Our HDX-MS experiments thus highlighted the structural elements that are likely responsible for evolution of the coelenterazine-powered bioluminescence at alpha/beta-hydrolase fold enzymes. Based on our results, we hypothesize that the increased backbone dynamics and conformational plasticity of the protein fold allows binding of the bulky bioluminescent substrate, coelenterazine.Complementary computational molecular dynamics (MD) simulations were also applied to describe intrinsic motions of ancHLD-RLuc enzyme. Catalytically essential residues pointed out by our structural studies and MD simulations were then subjected to site-directed mutagenesis, and the corresponding mutants were characterized biochemically to verify their roles in the underlying biocatalysis. Collectively, our results thus shed new light on the evolution of the Renilla-type bioluminescence and are important for designing next-generation luciferins (coelenterazine derivatives) and luciferases with fine-tuned photonic properties.Results of the project have been published in high-impact journals, and presented on international conferences and workshops. In addition, information about the Ancestral project and its results have been presented also to the general public (e.g. Researchers' Night events and visits in grammar high schools). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This MSCA project generated advanced knowledge towards dissecting the molecular basis and evolution of the coelenterazine-powered bioluminescence at alpha-/beta-hydrolase fold enzymes. Complementary biochemical and biophysical experiments provided unprecedented insights into the reaction mechanism underlying the light-emitting biocatalysis by Renilla-type luciferases, which significantly expanded our current understanding of the bioluminescent systems beyond the state-of-the-art. We expect that this newly gained knowledge will enable rational engineering of novel luciferases and luciferins with fine-tuned photonic properties. Precisely, the Ancestral project provided know-how for the design of coelenterazine-utilizing luciferases with alpha-/beta-hydrolase fold and displaying novel photophysical features. In addition, we anticipate that structural information on ancestral Renilla-type luciferases will help to design new coelenterazine derivatives, which will expand the toolbox with luciferins. Collectively, the project meaningfully contributed to: (i) our fundamental understanding of the molecular function and evolution of coelenterazine-powered Renilla-type bioluminescence, and (ii) our knowledge how to intelligently engineer next-generation of luciferases and luciferins for bioimaging and biosensor technologies. The biotech-oriented outcomes of this project have a potential to boost the development of superior bioluminescent systems applicable in biomedical technologies, which could globally and positively impact public health and well-being. Comparison of standard Renilla-type luciferase with a new engineered luciferase variant.