In vitro reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum

Pyruvate dehydrogenase (PDH) and 2‐oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum , an unusual hybrid complex consisting of Cg E1p (thiamine diphosphate‐dependent pyruvate dehydrogenase, AceE), Cg E2 (dihydrolipoamide acetyltransferase, AceF), Cg E3 (dihydrolipoamide dehydrogenase, Lpd), and Cg E1o (thiamine diphosphate‐dependent 2‐oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH‐ODH hybrid complex in C .  glutamicum probably consists of six copies of Cg E2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty‐four copies of E2. We found that Cg E2 formed a stable complex with Cg E3 ( Cg E2‐E3 subcomplex) in vitro , hypothetically comprised of two Cg E2 trimers and four Cg E3 dimers. We also found that Cg E1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the Cg E2‐E3 subcomplex. Our in vitro reconstituted system showed Cg E1p‐ and Cg E1o‐dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both Cg E1p and Cg E1o associate with a single Cg E2‐E3. In gel filtration chromatography, all the subunits of Cg ODH were eluted in the same fraction, whereas Cg E1p was eluted separately from Cg E2‐E3, suggesting a weak association of Cg E1p with Cg E2 compared with that of Cg E1o. This study revealed the unique molecular architecture of the hybrid complex from C .  glutamicum and the compact‐sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.