When enzymes join forces: unmasking a mitochondrial biosynthetic engine

Introduction

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 basis 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.

Biosynthesis of Coenzyme Q

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 synthesize 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 adhere to the inner-mitochondrial membrane. This infrastructure can extract the substrate whilst providing a shielded, hydrophobic environment for molecular transit.

Research Objectives

We will systematically characterize the functional, structural, and evolutionary aspects of the involved protein machineries in interplay with the membrane. Our approach includes the following steps:

1. Exploiting ancestral sequence reconstruction to generate proteins of enhanced stability.
2. Building the metabolon in vitro to assess how the enzymatic activities are coupled in the context of a metabolon.
3. Conducting structural studies to reveal how the active sites are spatially organized with respect to the order of the enzymatic steps and substrate trafficking.

Expected Outcomes

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 fueled and governed through interactions between enzymes, and between enzymes and other proteins.