High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Abstract

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.