Abstracts

The rational engineering of luciferase enzymes towards ultrasensitive bioassays is often possible only when the underlying catalytic mechanism is thoroughly known. Thus, atomic-level knowledge of a Michaelis enzyme-substrate complex, revealing molecular details of luciferin recognition and catalytic chemistry, is crucial for understanding and then rationally improving bioluminescent reactions. However, many known luciferase enzymes sample huge protein conformational space, often preventing complete structural characterization by X-ray crystallography. Moreover, using a cognate luciferin is problematic since its conversion into an oxyluciferin in the presence of the luciferase will prevent the capture of the enzyme-luciferin conformation in an activated state. Here, we outline how to deal with such obstacles, focusing on the recent discovery of a Renilla-type bioluminescence mechanism facilitated by a combination of engineered ancestral enzyme and the availability of a non-oxidizable luciferin analogue. The automated ancestral sequence reconstructions using FireProt^ASR provided an evolvable thermostable enzyme suited for structural studies [1], and a stable luciferin analogue azacoelenterazine provided a structurally cognate chemical incapable of catalyzed oxidation [2]. We suggest that an analogous strategy can be applied to various luciferases with unknown catalytic mechanisms and poor crystallizability [3].References [1] A. Schenkmayerova, G. P. Pinto, M. Toul, M. Marek, L. Hernychova, J. Planas-Iglesias, V. Daniel Liskova, D. Pluskal, M. Vasina, S. Emond, M. Dorr, R. Chaloupkova, D. Bednar, Z. Prokop, F. Hollfelder, U. Bornscheuer, J. Damborsky (2021) Nature Communications 12 3616.[2] A. Schenkmayerova, M. Toul, D. Pluskal, R. Baatallah, G. Gagnot, G.P. Pinto, V.T. Santana, M. Stuchla, P. Neugebauer, P. Chaiyen, J. Damborsky, D. Bednar, Y.L. Janin, Z. Prokop, M. Marek (2023) Nature Catalysis 6: 23-38.[3] T. Gao, J. Damborsky, Y.L. Janin, M. Marek (2023) ChemCatChem 15: e202300745.