A signal transduction cascade controlling 2-oxoglutarate dehydrogenase activity in the actinobacterial cell factory Corynebacterium glutamicum

A lowered 2-oxoglutarate dehydrogenase (ODH) activity is crucial for production of the flavor enhancer monosodium glutamate (MSG) by Corynebacterium glutamicum. ODH activity is inhibited by the regulatory protein OdhI in its unphosphorylated state. Phosphorylation of OdhI by the serine/threonine kinase PknG relieves inhibition. Previous studies indicated that the activity of PknG is controlled by the secreted putative glutamine-binding protein GlnH and the integral membrane protein GlnX.

The aim was to biochemically and structurally characterize the proteins GlnH and GlnX in order to obtain a detailed molecular understanding of their function. In addition, we aimed to analyze the cellular localization of OdhI and the pyruvate dehydrogenase (PDH)-ODH supercomplex.

The binding properties of purified GlnH were determined by ITC and tryptophan fluorescence quenching. The cellular localization of proteins was analyzed by fluorescence microscopy. Structural models of GlnH and GlnX were generated using the Colabfold2 pipeline by AlphaFold2.

GlnH binds L-aspartate and L-glutamate with moderate to low affinity, but not L-glutamine, L-asparagine, or 2-oxoglutarate. The predicted topology of GlnX with four transmembrane helices and two large periplasmic domains was confirmed by LacZ and PhoA fusions. Ab initio structure prediction of GlnX by AlphaFold2 revealed a novel architecture named as Helical Tandem Module (HTM) composed of three four-helix bundles (4HBs) – with one 4HB located in the membrane and two 4HBs in the periplasm. All subunits of the PDH-ODH complex were localized in distinct spots at the cell poles and at mid-cell, possibly due to nucleoid exclusion.

Our results provided novel information on the GlnH-GlnX-PknG-OdhI-ODH signal transduction cascade, which serves to adapt the carbon flux at the 2-oxoglutarate node between ammonium assimilation and energy generation via TCA cycle to the availability of L-glutamate and L-aspartate in the environment. Furthermore, an unexpected localization of the PDH-ODH was identified.