Abstract
Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (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 CgE2 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 CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. 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.