Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

Authors

Chaloupkova, R., Liskova, V., Toul, M., Markova, K., Sebestova, E, Hernychova, L., Marek, M. Pinto. G. P., Pluskal, D., Waterman, J., Prokop, Z., Damborsky, J.

Source

ACS CATALYSIS 9: 4810–4823 (2019)

Abstract

To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. The crystal structure revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants, and the molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen-deuterium exchange mass spectrometry. The stopped-flow analysis revealed that the alkyl-enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. In conclusion: a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at enzyme class-level, and ancestral reconstruction has a clear potential for obtaining multi-functional catalysts.

Full text •  Supplement

Citation

Chaloupkova, R., Liskova, V., Toul, M., Markova, K., Sebestova, E, Hernychova, L., Marek, M. Pinto. G. P., Pluskal, D., Waterman, J., Prokop, Z., Damborsky, J., 2019: Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts. ACS Catalysis 9: 4810–4823.

Gaspar a Gaussian Expert
1st Hands-on Computational Enzyme Design Workshop
Marie Curie Fellowship for Dmitri
Rayyan Announced as Best Presenter
Researchers’ Night 2019 in LL
Open Postdoctoral Position: Protein Structural Biologist
Andrea Awarded for Excellent Research