Furthermore, the sequence of AcmB also differs from the only structurally characterized representative of the KstDs , namely the KstD1 from Rhodococcus erythropolis (PDB: 4C3X and 4C3Y) . Our bioinformatic analysis indicates that KstD1 from R. erythropolis (GenBank accession code AAF19054.1) is shorter by approximately 50 amino acids when compared to the majority of the know KstD sequences. The longest difference in the amino acid sequence, 38 amino acids (compared to the sequence of AcmB, see Figure ), was previously referred to as the 'loop' . As a result, the crystal structure of KstD1 from R. erythropolis represents only a fraction of the known enzymes in the KstD family. In this work, we structurally characterize KstD from S. denitrificans (AcmB, GenBank accession code ABV59992), which, based on its amino acid sequence composition, represents most of the known KstDs. The structural characterization is combined with kinetic and modelling studies addressing the reaction mechanism with several steroid substrates (Figure ). With pre-steady-state kinetics, we explained the surprising origin of two pH optima of AmcB . We have also successfully used site-directed mutagenesis and QM/MM MD calculations to quantitatively explain the influence of active site residues on reaction kinetics. Finally, we discussed the biological role of the 'loop'.