Theoretical predictions of ∆Gb obtained for AcmB turned out to be similar to those published for KstD1 (Table -S6). The estimated free energy of binding for the effective enzymesubstrate complexes (G) for all analysed substrates ranged between -10 and -3.4 kcal/mol (Table ), but the Gs of the substrates with a degraded C17 substituent were in the range of -7.6 to -3.7 kcal/mol. The best-bound substrate turned out to be cholest-4-en-3-one (-10 kcal/mol), while diosgenone was bound only with G of -3.4 kcal/mol. The ΔGbest calculated for the 5 nanoseconds of the best E:S geometries led to similar conclusions (range from -8.7 to -6.1 kcal/mol, -12.4 kcal/mol for CHON). The analogical analysis conducted for the KstD1 from R. erythropolis yielded values of Gb in the range of -9.3 to -3.8 kcal/mol. Cholest-4-en-3-one was not different from other substrates (Gb of -6.3 kcal/mol), but diosgenon exhibited better stabilization than for AcmB (-5.8 kcal/mol). These results confirm the preferential binding of cholest-4-en-3-one by the enzyme with an additional 'loop' and explain the better apparent activity of KstD1 with diosgenone compared to AcmB . This result also suggests that the membrane-associated domain plays a role in the differentiation of substrates with extended C17 substituent (i.e., preferential binding of CHON over DSG).