Deciphering Enzyme Mechanisms with Engineered Ancestors and Substrate Analogues

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Authors

GAO Tadeja DAMBORSKÝ Jiří JANIN Yves L. MAREK Martin

Year of publication 2023
Type Article in Periodical
Magazine / Source ChemCatChem
MU Faculty or unit

Faculty of Science

Citation
Web https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202300745
Doi http://dx.doi.org/10.1002/cctc.202300745
Keywords ancestral sequence reconstruction; azacoelenterazine; biocatalysis; bioluminescence; coelenterazine; luciferase; enzymes; FireProt ASR; reaction mechanism; substrate analogues
Attached files
Description Environmentally friendly industrial and biotech processes greatly benefit from enzyme-based technologies. Their use is often possible only when the enzyme-catalytic mechanism is thoroughly known. Thus, atomic-level knowledge of a Michaelis enzyme-substrate complex, revealing molecular details of substrate recognition and catalytic chemistry, is crucial for understanding and then rationally extending or improving enzyme-catalyzed reactions. However, many known enzymes sample huge protein conformational space, often preventing complete structural characterization by X-ray crystallography. Moreover, using a cognate substrate is problematic since its conversion into a reaction product in the presence of the enzyme will prevent the capture of the enzyme-substrate conformation in an activated state. Here, we outlined how to deal with such obstacles, focusing on the recent discovery of a Renilla-type bioluminescence reaction mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non-oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProtASR provided a thermostable enzyme suited for structural studies, and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation. We suggest that an analogous strategy can be applied to various enzymes with unknown catalytic mechanisms and poor crystallizability.
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