BACKGROUND: 3-ketosteroid-Delta(1)-dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2-desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3-ketosteroid-Delta(1)-dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3). RESULTS: KsdD4 exhibited optimal activity at pH 7.0 and 35 degrees C. KsdD4 had the highest activity toward 4-androstene-3,17-dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure-based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate-binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side-chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed-batch bioconversion using resting cells resulted in efficient production of 2.66 g L-1 androst-1,4-diene-3,17-dione (ADD). CONCLUSION: Biochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed-batch strategy improved the bioconversion of steroids. (c) 2018 Society of Chemical Industry