Origin of functionality in nucleotide-dependent enzymes: the build-up of a complex catalytic network

YANG G; BAILLEUL G; NICOLL CR; MATTEVI A; FRAAIJE MW; MASCOTTI ML

Simposio; Gordon Research Seminar: Molecular Mechanisms in Evolution; 2023

Nucleotide-dependent enzymes are crucial for all life forms. These enzymes stand out from others because of two reasons, (i) they enable catalysis by the orchestrated action of a variety of elements –an electron donor/acceptor, a substrate, the amino acids at the active site- and, (ii) nucleotides were likely the drivers of the transition from chemical catalysis to enzymatic catalysis, a major contribution for life emergence on Earth. One of the main groups of extant, nucleotide-based cofactors are the flavins. Flavins allow cells to handle high energy electrons to oxidize/reduce metabolites and xenobiotics or to even generate energy gradients across membranes. Over the last years we have investigated the evolutionary history of the flavoprotein monooxygenases. This enzyme family comprises three well-defined, functionally-divergent groups: the FMOs, NMOs and BVMOs. To explore the origin and divergence of the functionality across the different groups, we resurrected and characterized ancestors from different branches and periods in the time-scale of life on Earth. First, we investigated the so-called flavin-containing monooxygenases (FMOs). We discovered that the functional divergence of nowadays jawed vertebrates FMOs is the result of functional optimization via gene-duplication, linked to the emergence of tetrapods (≈ 350 mya). A complex catalytic network involving epistasis relayed by the ligand allows some FMOs to be promiscuous towards non-canonical enzymatic activities as the BV oxidation. More recently, we focused on the prokaryotic flavin-containing Baeyer-Villiger monooxygenases (BVMOs). We followed a specific lineage of well-characterized extant enzymes across the great oxygenation event (≈ 2.3-2.5 bya). By deeply scanning the biochemical features of ancestral BVMOs we have been able to understand how the redox activity was first built by coenzyme recruiting and then followed with the monooxygenase-functionality optimization. Combined together, our results portrait how complex enzymatic mechanisms arose in evolution.