3-Ketosteroid Δ 1 -dehydrogenase (KsdD) is the key enzyme responsible for Δ 1 -dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.

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