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. 2015 Dec 30;4:e05322. doi: 10.7554/eLife.05322

Figure 3. S200 is critical for YcgC’s deacetylation activity.

(a) Two mutants of YcgC, that is, S S8/10/73/77/200A and S200A were constructed through gene synthesis. In vitro assays of the KDAC activity of these two mutants on RutR demonstrated that their KDAC activities were completely abolished. (b) S200 on YcgC was identified as an AEBSF binding site by LC-MS/MS analysis. YcgC was incubated with AEBSF, then trypsin digested and subjected to MS/MS analysis. Upon AEBSF-mediated sulfonation, a 183 Da molecular weight increase is predicted. (c) In vitro assays of the KDAC activity of YcgC on heat-denatured RutR demonstrated that YcgC was still active (right panel), while the downshift band disappeared (left panel). Similar results were observed when heat-denatured RutR was treated with CobB. KDAC: Lysine deacetylase; LC-MS/MS: Liquid chromatography–mass spectrometry; AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride; WT: Wild type.

DOI: http://dx.doi.org/10.7554/eLife.05322.010

Figure 3.

Figure 3—figure supplement 1. YcgC’s protein deacetylase activity is inhibited by AEBSF.

Figure 3—figure supplement 1.

(a) YcgC’s deacetylase activity on YcdC was monitored when a variety of hydrolase inhibitors were added individually. The solvents of these inhibitors, that is, DMSO and ethanol, were also included as controls. (b) Testing the individual component of the Pierce Halt protease inhibitor cocktail revealed that AEBSF inhibits the protein deacetylase activity of YcgC. (c) YcgC’s protein deacetylase activity is not affected by both EDTA and EGTA.
DOI: http://dx.doi.org/10.7554/eLife.05322.011
Figure 3—figure supplement 2. Lysine 62 is critical for RutR proteolysis.

Figure 3—figure supplement 2.

(a) Mutant proteins of K52R, K62R, K52/62R, K52Q, K62Q, and K52/62Q with WT, RutR proteins were treated with YcgC and examined with Coomassie stain. (b) The ratio of cleavaged RutR to intact RutR was also calculated.
DOI: http://dx.doi.org/10.7554/eLife.05322.012
Figure 3—figure supplement 3. The Km and Vmax values of YcgC were determined using RutR as a substrate.

Figure 3—figure supplement 3.

Because the N-terminal cleavage of RutR is tightly coupled with its deacetylation by YcgC, the downshifted band of RutR in the YcgC deactylation reaction could be conveniently used as a surrogate of YcgC’s activity. YcgC was incubated with serially diluted acetylated RutR. Deacetylation was determined by measuring the intensity of the lower bands on a silver-stained gel.
DOI: http://dx.doi.org/10.7554/eLife.05322.013