MetaQ · When enzymes join forces: unmasking a mitochondrial biosynthetic engine

Enzymes have been classically investigated as standalone catalysts operating in a relatively diluted milieu. However, the cell micro-compartments are highly crowded environments and biological catalysis cannot be fully understood on the bases of simple diffusive models. We are tackling this challenge by reconstituting a full-scale biosynthetic pathway where multiple enzymes coordinate within a metabolon - a structurally defined setting that allows the vectorial transfer of substrates and products.Our system for exploration is the fascinating biosynthesis of coenzyme Q, an essential redox mediator for many pathways. The juxtaposition between its highly polar head group and hydrophobic tail renders this compound a challenging feat to handle. To synthesise its highly substituted aromatic head group, nature has amassed a large soluble supra-molecular complex consisting of no less than eight functionally distinct proteins that adheres to the inner-mitochondrial membrane. This infrastructure can extract the substrate whilst providing a shielded, hydrophobic environment for molecular transit.We will systematically characterize the functional, structural and evolutionary aspects of the involved protein machineries in interplay with the membrane. Our approach starts by exploiting ancestral sequence reconstruction to generate proteins of enhanced stability. We will build the metabolon in vitro to assess how the enzymatic activities are coupled in the context of a metabolon. Structural studies will reveal how the active sites are spatially organized with respect to the order of the enzymatic steps and substrate trafficking. Our integrated strategy will unveil the pivotal evolutionary transitions that create a biosynthetic machinery. This research will go beyond classical enzymology by exploring a new paradigm of cellular biochemistry where metabolic pathways are fuelled and governed through interactions between enzymes, and between enzymes and other proteins.