Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein.
A novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the delta pknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a delta odhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a Ki value of 2.4 nM. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle.
Niebisch
Axel
A
Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Kabus
Armin
A
Schultz
Christian
C
Weil
Brita
B
Bott
Michael
M
D000595:Amino Acid Sequence; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D016680:Genome, Bacterial; D005973:Glutamine / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism; D014233:Tricarboxylic Acids / Q000378:metabolism
D010750:Phosphoproteins; D014233:Tricarboxylic Acids; D005973:Glutamine; D013912:Threonine; D007655:Ketoglutarate Dehydrogenase Complex; D017869:Cyclic GMP-Dependent Protein Kinases