The secondary structure of the putative membrane-associated domain, a combination of sheets and -helices, turned out to be much more complex than previously predicted by homology modelling . The bioinformatic analysis of KstD sequences with known biological functions shows the prevalent presence of this domain in the enzyme class. Furthermore, the sequences differ significantly, forming separate clades. Although the data on the substrate specificity of the representatives of each clade is still not plentiful, we suspect that the differences in the sequence of the membrane-associated domain are partially responsible for the reported differences in the substrate specificity. Enzymes belonging to the ReKstD1-like clade all lack a membrane-associated domain and are reported to be inactive or slightly active with C17-substituted steroids . We already demonstrated that ReKstD1 converts cholest-4-en-3on if proper solubilization is provided. Still, ReKstD1 has a definitively higher preference for smaller substrates. Until now, the only characterized members of AcmB and AmcB2-like clads are AcmB and AcmB2, which are both capable of converting C17-substituted substrates, although with different structural characteristics . Moreover, KstD3 from P. simplex, a representative of the PsKstD3-like clade, has been reported as active with cholest-4-en-3-one along with several other isoenzymes for which this activity was not observed. Interestingly, a similar phylogenetic tree can be obtained when only sequences of the membrane-associated domain are taken into consideration (Figure ). As a result, we shall assume that the sequence of the putative membrane-associated domain strongly influences the divergence of the KstD family and may be responsible for the different biological roles of particular enzymes.