Fig. 2

AncCARs have CAR-like substrate kinetics. a Turnover of NADPH by AncCARs was measured with 24 unique carboxylic acids, of which 5 are shown here. Bar chart shows activity on canonical acid substrates at 5 mM. Each substrate was tested using three experimental replicates. Asterisks represent degrees of significance from t-test of each experiment compared to controls (* = 0.0001 < P ≤ 0.001; **0.00001 < P ≤ 0.0001; *** = 0.000001 < P ≤ 0.00001; **** = P ≤ 0.000001). Complete substrate screens are presented in Supplementary Fig. 13. b The active site structure of the adenylation domain. AncCARs A (green), PA (blue), and PF (red) are overlaid onto S. rugosus CAR (PDB ID: 5MST; yellow). Substrates are shown in gray. The residues lining the active site pocket of ExCARs (positions 246–250) are poorly resolved. c In AncCAR-PF, the highly variable loop between positions 286 and 302 of the adenylation domain of AncCAR-PF (red) does not interact with the substrate, in contrast to SrCAR (5MST; yellow). Images produced in PyMOL v.2.2.