[Seminar] In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis

Title: In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis

Speaker: Andrea Mattevi

From: Department of Biology and Biotechnology, University of Pavia (Italy)

Coenzyme Q, CoQ, is an essential antioxidant ubiquitous in nature. CoQ biosynthesis in humans has thus far been attributed to ten different proteins with many coalescing into a large assembly coined the ‘COQ metabolon’. Metabolons are protein assemblies that channel substrates within a metabolic pathway, however, their general aptitude for enzymatic biocatalysis remains poorly understood and several CoQ biosynthetic steps, including C1-decarboxylation and C1-hydroxylation steps, remain enigmatic in our understanding. To elucidate the prerequisites for CoQ biosynthesis and moreover, assess the physicochemical properties of the metabolon, we sought out to construct the entire metabolon in vitro. We employed an innovative evolution-engineering approach rooted by ancestral sequence reconstruction, in addition to spectrophotometric and biophysical assays, and analytical techniques to monitor CoQ biosynthesis. We reconstructed tetrapod ancestors for COQ proteins: COQ3, COQ4, COQ5, COQ6, COQ7, COQ8A, COQ8B, COQ9 and Ferredoxin reductase and Ferredoxin 2, speculated to initiate CoQ biosynthesis.

We observed activity for all ancestral COQ proteins and demonstrated that COQ4 is a Zn-dependent decarboxylase responsible for the C1-decarboxylation and that COQ6 performs the subsequent C1 hydroxylation step. Amalgamating all the proteins together allowed us to construct the entire biosynthetic pathway in vitro. Furthermore, the formation of the COQ metabolon, mediated by an atypical kinase, COQ8B, increases CoQ production and reduces intermediate accumulation. Using a combination of HDX, XL-MS, microscale thermophoresis, and size exclusion chromatography techniques, we have been able to capture several binary interactions including COQ6:COQ3 and COQ7:COQ9 to name a few. Furthermore, using Native PAGE analysis and XL-MS, we have observed the formation of different assemblies with unique crosslinking networks, implying a dynamic and heterogeneous composition of protein interactions.
Our findings suggest that metabolons possess heightened catalytic efficiency, consist of an intricate and dynamic number of protein interactions, and are overseen by chaperoning and organisational protein mitigators.

References:
Nicoll, C.R., Alvigini, L., Gottinger, A. et al. In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis. Nat Catal 7, 148–160 (2024).