Ancestral · Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity

Structural and biochemical studies of an ancestral enzyme with dual dehalogenase and luciferase activity

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.

Data: CORDIS, © European Union