Metabolons are protein assemblies that perform a series of reactions in a metabolic pathway. However, the general importance and aptitude of metabolons for enzyme catalysis remain poorly understood. In animals, biosynthesis of coenzyme Q is currently attributed to ten different proteins, with COQ3, COQ4, COQ5, COQ6, COQ7 and COQ9 forming the iconic COQ metabolon. Yet several reaction steps conducted by the metabolon remain enigmatic. To elucidate the prerequisites for animal coenzyme Q biosynthesis, we sought to construct the entire metabolon in vitro. Here we show that this approach, rooted in ancestral sequence reconstruction, reveals the enzymes responsible for the uncharacterized steps and captures the biosynthetic pathway in vitro. We demonstrate that COQ8, a kinase, increases and streamlines coenzyme Q production. Our findings provide crucial insight into how biocatalytic efficiency is regulated and enhanced by these biosynthetic engines in the context of the cell.

代谢物是在代谢途径中执行一系列反应的蛋白质组装体。然而，代谢物对酶催化的一般重要性和能力仍然知之甚少。在动物中，辅酶Q的生物合成目前归因于十种不同的蛋白质，其中COQ3、COQ4、COQ5、COQ6、COQ7和COQ9形成了标志性的COQ代谢物。然而，代谢物进行的几个反应步骤仍然是个谜。为了阐明动物辅酶 Q 生物合成的先决条件，我们试图在体外构建整个代谢物。在这里，我们展示了这种植根于祖先序列重建的方法，揭示了负责未表征步骤的酶，并捕获了体外生物合成途径。我们证明 COQ8（一种激酶）可以增加并简化辅酶 Q 的产生。我们的研究结果为了解这些生物合成引擎在细胞中如何调节和增强生物催化效率提供了重要的见解。