(A) RutR AcK52 and RutR AcK62 were treated with a two-fold molar excess of YcgC, YcgC S200A, CobB or CobB H110Y in the presence of 5 mM NAD+. As visible from the immunoblotting using an anti-AcK AB CobB was able to completely deacetylate RutR AcK52 while RutR AcK62 is only faintly deacetylated under the conditions used. YcgC, YcgC S200A and the catalytically dead CobB H110Y did neither deacetylate RutR AcK52 nor RutR AcK62 suggesting that YcgC is no deacetylase for RutR. We used the GST-fusion proteins of CobB and CobB H110Y here to get a better separation of CobB and RutR proteins. Probing of the His6-tag using an anti-His6 antibody was done to show equal loading with RutR proteins. SDS-PAGE analysis and CBB staining show that there is only one band of the same size for RutR for all the conditions tested suggesting that YcgC did also not stimulate (auto-) proteolytic cleavage of RutR. The control lane (ctrl) shows the respective RutR protein, AcK52 or AcK62, without addition of CobB or YcgC. Molecular weights of proteins used (all masses calculated without N-terminal methionine): RutR, 24567.5 Da; acetylated RutR, 24609.5 Da; GST-CobB, 53271.09 Da; GST-CobB H110Y, 53297.12 Da.; YcgC, 51649.80 Da; YcgC S200A, 51633.80 Da. We used the GST-fusion proteins for CobB to obtain a better separation from RutR as CobB (MW: 26314.86 Da) and CobB H110Y (26340.90 Da) without GST-tag have similar molecular weights compared to RutR. (B) Quantification of ESI-MS spectra of YcgC and CobB reaction products shown as immunoblots in (A). As a support for the data obtained by immunoblotting, neither YcgC nor YcgC S200A nor catalytically inactive CobB H110Y did alter the molecular mass of RutR WT, AcK52 and AcK62. However, active CobB led to more than 60% deacetylation of RutR AcK52 while RutR AcK62 was only marginally deacetylated (app. 30% deacetylated). Again, these data confirm that YcgC does neither deacetylate or proteolytically cleave RutR nor does it stimulate autoproteolytic cleavage of RutR.