An extant enzyme with dual dehalogenase‒luciferase function

M. Majerova1,2, M. Vasina1, D. Pluskal1, J. Krizkova1, J. Damborsky1,2, Z. Prokop1,2, J. Delroisse3, M. Marek1,2

1Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

2International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 65691 Brno, Czech Republic

3Biology of Marine Organisms and Biomimetics Unit, University of Mons, Pentagone 2B, 6 Avenue du Champ de Mars, 7000 Mons, Belgium

marika.majerova@recetox.muni.cz

Haloalkane dehalogenases (HLDs, EC 3.8.1.5) are mostly microbial α/β-hydrolase fold enzymes that cleave carbon-halogen bonds in diverse halogenated hydrocarbons. Interestingly, these enzymes exhibit significant sequence and structural similarity with Renilla-type luciferases (EC 1.13.12.5). These luciferases are decarboxylating monooxygenases that produce blue light through the oxidation of a luciferin (coelenterazine) [1,2]. Here, we identified several genes coding for putative dehalogenase/luciferase-like enzymes in the draft genome of Amphiura filiformis [3], a luminous brittle star. Protein expression trials showed that some of these putative enzymes are expressed as soluble and properly folded proteins in Escherichia coli. Surprisingly, our biochemical experiments showed that one enzyme exhibits a dual function, catalyzing the hydrolytic dehalogenase reaction and the oxidation of coelenterazine followed by the emission of blue photons. Co-crystal structures reveal an as-yet-unseen mode of luciferin binding, which is radically dissimilar to that observed in Renilla luciferase and suggests a non-canonical, less-effective coelenterazine oxidation. Structure-based mutagenesis experiments support this unusual mode of catalysis. Our findings collectively support the hypothesis that coelenterazine-powered Renilla-type luciferases evolved from HLD-fold proteins.

1. Schenkmayerova A., Toul, M., Pluskal D., Baatallah R., Gagnot G., Pinto G. P., Santana V. T., Stuchla M., Neugebauer P., Chaiyen P., Damborsky J., Bednar D., Janin Y. L., Prokop Z., Marek M. (2023). Catalytic mechanism for Renilla-type luciferases. Nature Catalysis 6, 23-38, https://doi.org/10.1038/s41929-022-00895-z

2. Delroisse J., Duchatelet L., Flammang P., Mallefet J. (2021). Leaving the dark side? Insights into the evolution of luciferases. Frontiers in Marine Science 8, 690, https://doi.org/10.3389/fmars.2021.673620

3. Buckley K., Delroisse J., Ortega-Martinez O., Oliveri P., Martelaz F., Martinez P., Parey E., et al. Genome consortium working on the genome of A. filiformis (unpublished data).

This work was supported by the Czech Science Foundation (22-09853S).