Fig. 7.

Succinylation profiling of Acs of Mtb. A, Schematic illustration of regulation of Acs activity by reversible acetylation and succinylation. B, Identification of the modification sites of Acs. The representation of the corresponding MS/MS spectra of succinylated peptides from Acs annotated with a comprehensive series of b and y fragment ions. Details of succinylation sites were listed in the supplemental Table S1. C, The acetylation/succinylation levels of purified recombinant Acs were determined by immunoblotting using specific anti-acetyl/succinyl lysine antibodies. D, Succinylation of Lys193 and Lys366 affect the enzyme activity of Acs. The Acs and its mutants (Acs-K193R, Acs-K366R, and Acs-K193R/K366R) were expressed in E. coli BL21 (DE3) and the specific activity of Acs was determined. Data are means ± S.D. from three independent assays. E, NAD⁺-dependent deacetylation/desuccinylation of Acs by CobB protein (Rv1151c). Acs was incubated with CobB in the presence of NAD⁺ (0.5 mm) for 10h at 25 °C. Nicotinamide (NAM, 2 mm) was added to the reaction as CobB inhibitor. F, Quantification of acetylated/succinylated Acs in the reactions from E. Each sample was standardized by comparing to the Coomassie blue stained gel. Error bars indicate S.D. of three measurements.