Deciphering Enzyme Mechanisms with Engineered Ancestors and Substrate Analogues
| Autoři | |
|---|---|
| Rok publikování | 2023 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | ChemCatChem |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202300745 |
| Doi | http://dx.doi.org/10.1002/cctc.202300745 |
| Klíčová slova | ancestral sequence reconstruction; azacoelenterazine; biocatalysis; bioluminescence; coelenterazine; luciferase; enzymes; FireProt ASR; reaction mechanism; substrate analogues |
| Přiložené soubory | |
| Popis | 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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