The modelling showed that the energy profile for AD is very similar to that of 17-MT (Figure ). This was expected because the only structural difference between AD and 17-MT (substitution of C17) is pointed toward the entrance to the active site (Figure ). However, in the case of DHT, which has no double bond in ring A, we observed an elevation of both barriers and energy of E:I by approx. 3 kcal/mol. The saturated A ring of the substrate results in a slightly later TS1 and an earlier TS2 compared to the geometries obtained with AD (Figures and) and higher charge separation in the E:I. These results are consistent with our steady-state kinetic experiments, which yielded respectively 71% and 19% specific activity of AcmB with 17-MT and DHT with respect to AD (Table ). Interestingly, we obtained the best PES for AcmB native substrate (Fig. ), CHON. The C-H activation was very similar to AD (Fig. ), yielding only a slightly lower barrier (12.8 kcal/mol). However, the hydride transfer to FAD proceeds with a barrier of only 14.1 kcal/mol. This result suggests that the RHR for CHON should proceed with the highest rate of all investigated substrates. Unfortunately, due to the low water solubility of CHON, it was not possible to directly compare the AcmB dehydrogenation rate of CHON and AD .