Tracking the evolution of the enzyme function in flavin-containing monooxygenases

MASCOTTI ML; BAILLEUL G; NICOLL C; MATTEVI A; FRAAIJE MW

Conferencia; XXIII NCCC, The Netherlands' Catalysis and Chemistry Conference; 2022

Flavin-dependent monooxygenases (FMOs) form a widespread enzyme family. In animals, they areinvolved in the detoxification of xenobiotics. Typically, they catalyze the oxygenation oxidation ofheteroatom-containing molecules, such as sulfides and amines. In the human proteome there arefive different FMOs, named FMO1-5. These differ in their tissue and developmental expressionpatterns. However, the most striking difference is in the reaction they catalyze: FMO1-3 carry outthe oxidation of heteroatom-containing molecules, while FMO5 catalyzes the Baeyer-Villiger (BV)oxidation of ketones. The origin of this atypical behavior is explored here by means of a historicalbiochemistry approach using ancestral sequence reconstruction (ASR). ASR allows inferring thesequences of the most likely ancestors of a protein family using as input the sequences of extantproteins. The ancestors are later expressed and characterized in the lab. This way, thedeterminants of enzyme function can be disclosed and therefore, ASR becomes an immenselyvaluable tool to engineer enzymes with biocatalytic purposes.Animal FMOs diversified from a single common ancestor in coincidence with the emergenceof tetrapods on earth. Previously, we successfully reconstructed and characterized the fourmammalian ancestors of FMOs: mAncFMO1, 2, 3 and 5. We were able to understand the specificbiochemical and structural features of each of them . Here, we tackled the reconstruction of thepre-diversification ancestor in early tetrapods tAncFMO1-5, and the two post-diversificationancestors: AncFMO1-4 and AncFMO5 (Fig. 1A). The tAncFMO1-5 was found to behave as abifunctional enzyme catalyzing heteroatom-containing molecules and BV oxidations. Interestingly,the tAncFMO1-4, completely lost the ability to catalyze BV oxidations (Fig. 1B). Further mutationalanalyses were performed to find the group of residues defining the BV activity. This provides agood view on the evolution of the different FMOs and how their catalytic properties diversified intime.