α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

ABSTRACTα-ketoacid dehydrogenase complexes are one the biggest enzymatic complexes found in nature with molecular mass ranging from several to more than 10 mega Daltons. This tripartite enzymatic machinery carries out key reactions in central metabolism. With an architecture extremely conserved, they have so far all considered to be structured around a hollow, highly symmetric core made by up to 60 subunits of the acyltransferase component. By using an integrative structural biology approach, we provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, due to a unique C-terminus structure that has a three-residue insertion. This insertion was found in organisms over the whole Actinobacteria class. We show how this insertion affects the oligomerization properties and the whole three-dimensional architecture of the acetyltransferase AceF, proposed to serve as the central component of a mixed supercomplex that bears together the pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) activities. As a counterexample, we also show how the acyltransferase component of the branched-chain ketoacid dehydrogenase (BCKDH) complex from Mycobacterium tuberculosis does not possess such insertion, and shows a more canonical cubic architecture. This new architecture challenges the widely accepted paradigm for 2-oxoacid dehydrogenases, based in all available structures from Eubacteria, Archaea and Eukaryotes, and that was assumed to be universally conserved. Our results shed new light on the structure and evolution of α-ketoacid dehydrogenase complexes involving one of the biggest phyla in Eubacteria, a phylum that includes species relevant for biotechnology as well as many human pathogens. Moreover, components of this PDH/ODH supercomplex are key for M. tuberculosis survival in the human host, and its unique core and protein-protein interactions represent potentially “druggable” targets.

No abstract