We determined the structure of KstD from S. denitrificans (AcmB) demonstrating for the first time the structure of the so-called 'loop' which is a characteristic motif for the majority of the known KstD sequences. This putative membrane-associated domain may be responsible for anchoring the enzyme to the cytoplasmic membrane, positioning the enzyme active site toward the source of the substrate as well as stabilization of the E:S complex with C17-substituted 3ketosteroids. With QM/MM MD modelling and kinetic studies, we confirmed that the 1,2dehydrogenation catalysed by AcmB proceeds according to the Ping-Pong bi-bi mechanism, while RHR according to the accepted two-step elimination mechanism. We showed that OHR, not RHR, is responsible for the low values of KIE observed in the steady-state experiments as well as peculiar pH optima. As in the case of KstD1 from R. erythropolis, the nature of FES is responsible for the KIE observed for both C1 and C2 labelled substrates, which once again demonstrates that our findings are general for KstD class. Our modelling also provided quantitative insight into the role of active site tyrosines, the influence of the  4 -double bond in the steroid on its activity and corroborated experimental evidence on non-obligatory enantioselectivity during C-H activation.