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).
