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
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.
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).