Fig. 6. CobB regulates ENO activity and influences cell growth by erasing Khib and Kac.

(A) Multiple sequence alignment of ENO from different species by Clustal×2. (B) Identification of K326ac and K343hib in ENO. (C) Detection of the activity of ENO WT and its mutants (K343T, K326Q, and K326Q/K343T) (data are means ± SEM from three independent assays; *P < 0.05, **P < 0.001). (D) Western blotting determined the Khib, Kac, and Ksucc levels of purified recombinant ENO. (E) Western blotting revealed that NAD-dependent CobB can catalyze de-2-hydroxyisobutyrylation/deacetylation/desuccinylation with ENO. ENO was incubated with CobB in the presence of NAD⁺ (0.5 mM) for 10 hours at 25°C. NAM (10 mM) was added to the reaction as a CobB inhibitor. (F) LC-MS detection of lysine de-2-hydroxyisobutyrylation and deacetylation activities of CobB with ENO peptides [GIANSILI-K(hib)FNQIGSLTETLAAIK, IQLVGDDLFVTNTK(Ac)ILK]. (G) Comparison of the quantities of K326ac and K343hib in T vector and CobB-overexpressing P. mirabilis (data are presented as means ± SEM, and P values were determined by two-tailed Student’s t test; NS, not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). (H) Measurement of the PEP amounts of T vector and CobB-overexpressing P. mirabilis (data are presented as means ± SEM; P values were determined by two-tailed Student’s t test; **P < 0.01). (I) Measurement of the PEP amounts of ENO-overexpressing P. mirabilis that transformed ENO WT and mutation (K326Q, K343T, and K326Q/K343T) vectors (data are means ± SEM from three independent assays; *P < 0.05, **P < 0.001). (J) Measurement growth curve of ENO-overexpressing P. mirabilis that transformed ENO WT and mutation (K326Q, K343T, and K326Q/K343T) vectors (data are means ± SEM from three independent assays, *P < 0.05). OD₆₀₀, optical density at 600 nm. (K) Graphic model as discussed in the text. CobB can promote bacterial growth by effective de-2-hydroxyisobutyrylation of K343 and deacetylation of K326 of ENO. 2PG, 2-phosphoglycerate.