As expected, the substrate specificity is not solely controlled by the putative membraneassociated domain but by the structure of the active site. We have observed differences in G binding even between steroid substrates with relatively similar structures. The sequence alignment analysis of 82 KstDs, as well as the previous report , revealed that many of the residues in the 5 Å radius of the steroid ligand are highly conserved among all clads of the KstDs. The anchoring motive of the 3-ketosteroid group ( 536 YhGhG 540 ) is highly conserved as well as catalytically critical Y363 and Y118. The rest of the tyrosine residues forming the proton relay systems are less conserved, with Y115, Y466 and Y467 present in 75, 68 and 69% of the analysed sequences, respectively. Interestingly, the substitution of Y466 and Y467