Fig. 5: The effect of cAMP on Sg 2-MIBS activity and cAMP binding to Sg Enc CBD.

a In vitro activity assays of 2-MIBS-loaded Sg Enc in the absence and presence of cAMP. Free 2-MIBS activity is shown for reference. k_cat/K_m: catalytic efficiency of 2-MIBS. Data are shown as mean values, with error bars representing the standard deviation of three independent experiments. Source data are provided as a Source Data file. b GC-MS analysis of 2-MIBS activity assays. All assays yielded 2-MIB as the main product, with its diagnostic mass fragment (m/z + 95) identified as the most abundant ion¹⁴. A 2-MIB standard is shown for reference (top). Source data are provided as a Source Data file. c Differential scanning fluorimetry (DSF) thermal melts of 2-MIBS-loaded Sg Enc in the absence or presence of cAMP. T_m: melting temperature. Source data are provided as a Source Data file. d 2-MIBS-loaded Sg Enc binding to cAMP-modified agarose resin. A representative SDS-PAGE gel is shown demonstrating that 2-MIBS-loaded Sg Enc is unable to bind to cAMP-modified agarose resin. The positive control Ec CAP is able to bind to the resin. Load: sample loaded onto the resin. 20 mM cAMP (orange): elution from the resin with 20 mM cAMP. This experiment was repeated independently four times. Source data are provided as a Source Data file. e Cryo-EM analysis of Sg Enc in the presence of cAMP. No significant conformational changes of the CBDs or the HK97-domain could be observed when compared to cryo-EM maps obtained in the absence of cAMP. Improved CBD density was obtained via 3D classification followed C1 homogeneous refinement, but no conformational changes could be observed. f Cryo-EM density of the β-barrel CBD binding site is shown (top). No additional density could be observed in the presence of cAMP. The metal-binding subdomain (MBD) is shown in purple. Structural model of the binding site with binding site residues shown as stick (bottom).