Although we were only able to verify these calculations with steady-state kinetics, which revealed a 5-fold slower oxidation of DHT with respect to AD, we can assume that in the case of DHT, it is the RHR which predominantly controls the observed enzyme activity instead of OHR, as is the case with the other substrates which are oxidized faster. Therefore, we propose that this may explain the observed differences in KstD specificity toward solely  4 -3ketosteroid . The modelling also confirmed our previous kinetic analysis which pointed out at cholest-4-en-3-one as a native substrate . Not only is CHON preferentially bound by the enzyme (the lowest total and the best ∆G and IE), but also its RHR is associated with the lowest barriers for both C-H activation and H -transfer.