Effect of a C C double
Effect of a C4-C5 double bond — The presence of a double bond at the substrate’s C4-C5 position affects the reactivity of Δ1-KSTDs to varying extent, depending on the enzyme. Most 3-ketosteroids that are converted by Δ1- KSTDs have this double bond. For some Δ1-KSTDs this double bond is even required, such as the Δ1-KSTD from N. simplex ATCC 6946 and IFO 12069, which had no activity on 5α- (31) and 5β-androstane-3,17-dione (32) [48,51,52]. Similarly, both Δ1-KSTDs from wild-type M. fortuitum ATCC 6842 were able to 1(2)-dehydrogenate 9-OHAD (34), while they were inactive on 9α-hydroxy-5-androstene-3,17-dione (35) . On the other hand, the double bond was not necessary for the activity of a Δ1-KSTD from B. sphaericus ATCC 7055 since this enzyme 1(2)-dehydrogenated 5α-androstane-3,17-dione (31) and AD (8) with identical Vmax values . Likewise, a Δ1-KSTD from S. denitrificans Chol-1ST was active on 5-cholesten-3-one (59) with the same catalytic efficiency (kcat/KM) as on 4-cholesten-3-one (2) . Δ1-KSTD3 from R. erythropolis SQ1 and a Δ1-KSTD from M. tuberculosis H37Rv even preferred 5α-3-ketosteroids with a saturated A-ring such as 5α-androstane-3,17-dione (31), 5α-testosterone (23), and 5α-pregnane-3,20-dione (42). Intriguingly, these enzymes were seemingly inactive on AD (8) and several other Δ4-3-ketosteroids . Similarly, a Δ1-KSTD from the intestinal bacterium Clostridium paraputrificum was not able to 1(2)-dehydrogenate AD, but, in contrast to the former two enzymes, it was only active on 5β-3-ketosteroids like 5β-androstane-3,17-dione (32) and 3-oxo-5β-cholan-24-oic Cytochrome Oxidase Activity Colorimetric Assay Kit (57) . Thus, these data show that Δ1-KSTDs may differ considerably in their requirement for the presence of a double bond at the C4-C5 position of the steroid substrate. Substituents at the C9 position — Δ1-KSTDs respond also differently to 9α-substituted 3-ketosteroids. A Δ1-KSTD from R. equi 1(2)-dehydrogenated 9α-fluorocortisol (50) and 9α-fluoro-16α-hydroxycortisol (51) with comparable activities as cortisol (48), which has no substituent at C9 . Similarly, a Δ1-KSTD from N. simplex ATCC 6946 was not inhibited by the presence of a fluorine substituent at the 9α position . 9-OHAD (34), a key intermediate of microbial cholesterol degradation, which contains a hydroxyl group at C9, was converted by Δ1-KSTD1 and Δ1-KSTD2 from R. erythropolis SQ1 , as well as by all Δ1-KSTDs from M. fortuitum ATCC 6842  and M. neoaurum ATCC 25795 . In contrast, this steroid was not converted by Δ1-KSTD3 from R. erythropolis SQ1 and a Δ1-KSTD from M. tuberculosis H37Rv . Thus, while it appears that Δ1-KSTD1s can accept a small fluorine substituent at C9, a larger hydroxyl group cannot be accommodated by all enzymes. Substituents at the C10 position — A methyl group at the 10β position of 3-ketosteroid substrates is not essential for Δ1-KSTD function. The Δ1-KSTDs from C. testosteroni ATCC 11996 , R. equi , N. simplex ATCC 6946 and IFO 12069 [48,49,52], and S. denitrificans Chol-1ST  all catalyzed 1(2)-dehydrogenation of 19-nor-testosterone (22). Moreover, Δ1-KSTD from R. rhodochrous IFO 3338  was active on 19-nor-4-androstene-3,17-dione (28). Yet, these steroids were 1(2)-dehydrogenated less rapidly by the enzymes than their 10β-methyl counterparts [27,29,47,48,50]. The 10β-methyl group is possibly recognized by the enzyme, since in the crystal structure of Δ1-KSTD1•ADD (Protein Data Bank code 4c3y ) the 10β-methyl group is at van der Waals distance to the Phe-116, Phe-294, Tyr-318, and Ile-354 side chains. Interestingly, a Δ1-KSTD from Moraxella sp. was able to 1(2)-dehydrogenate a substrate with a hydroxymethyl group at the 10β position, i.e. 4-androsten-19-ol-3,17-dione (38)  and a Δ1-KSTD from N. simplex ATCC 6946 was reported to be active on 3-ketosteroids with an ethyl group at the 10β or 13β positions, but not with larger substituents at these positions . Indeed, inspecting the Δ1-KSTD1•ADD structure, enough space appears to be present in the active site of the enzyme to accommodate ethyl, but not larger substituents at the 10β or 13β positions.