A repurposed pseudoenzyme enables redox- and acetylation-dependent control of CoA transferase

Abstract

CoA transferases play essential roles in short-chain fatty acid and acyl-CoA metabolism, yet how their activities are regulated in response to metabolic signals remains unclear. Here, we show that a CoA transferase from Thermus thermophilus is controlled by a two-layer mechanism that integrates redox sensing with protein acetylation. The enzyme forms a complex with a catalytically inactive alanine dehydrogenase-like protein, a pseudoenzyme repurposed as an NAD+/NADH-ratio sensor. Biochemical and structural analyses revealed that NAD+ binding stabilized the alanine dehydrogenase-like protein in a conformation that promotes high-affinity association with the CoA transferase and restricts the flexibility required for catalysis, thereby imposing potent redox-dependent inhibition. Acetylation of the CoA transferase alleviates this NAD+-induced enzymatic inhibition, providing another regulation level dependent on the pool of intracellular acetyl-CoA levels. These findings establish CoA transferase as a previously unrecognized metabolic control point that senses the cellular NAD+/NADH ratio together with acetyl-CoA availability to fine-tune β-oxidation flux. Our work shows that a dual metabolite-sensing regulatory platform can emerge through the functional repurposing of a pseudoenzyme, expanding the current understanding of CoA transferase regulation. CoA transferases are crucial in fatty acid metabolism, yet their regulation by metabolic signals is not well understood. Here, the authors reveal a dual-layer regulatory mechanism in Thermus thermophilus, in which a pseudoenzyme mediates redox-dependent regulation of CoA transferase, while acetylation further modulates its activity.