Delta(1)-Dehydrogenation is one of the most important reactions for steroid drug modification. Numerous 3-ketosteroid-Delta(1)-dehydrogenases (KstDs) catalyzing this reaction were observed in various organisms. However, only a few have been characterized and used for substrate conversion. In this study, a promising enzyme (KstD2) from Mycobacterium neoaurum DSM 1381 was purified and characterized. Interestingly, KstD2 displayed a high activity on a range of substrates, including 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-OH-P); androsta-4,9(11)-diene-3,17-dione (NSC 44826); and 4-androstene-3,17-dione (AD). These reactions were performed under optimal conditions at 40 degrees C and pH 8.0. Noteworthy, both the activity and stability of the enzyme were sensitive to various metal ions. After optimizing the expression and biocatalyst conditions, up to 1586 U mg(-1) intracellular KstD activity on AD could be produced. Furthermore, the associated conversion rate was 99% with 30 g L-1 AD after 8 h. On the other hand, we obtained 99%, 90%, and over 80% of conversion with 20 g L-1 NSC 44826; 10 g L-1 16,17 alpha-epoxyprogesterone; and 20 g L-1 17 alpha-OH-P or canrenone, respectively, after 24 h. Sequence homology and structural analyses indicated that the residue R178 located in a unique short loop among cluster 2 is crucial for substrate recognition which was confirmed by mutagenesis. In summary, this study reports on the first purification and characterization of a KstD from cluster 2. Its remarkable properties deserve more attention to potentially lead to further industrial applications.