AP20187 Besides by substrate and product KSTDs may
Besides by substrate and product, Δ1-KSTDs may also be inhibited by other steroids. N. simplex ATCC 6946 Δ1-KSTD was strongly inhibited non-competitively by dicortinone (60), a steroidal dimer, and by bis-1-dehydrodicortinone, with values of 0.7 and 0.75 μM, respectively. This enzyme was also inhibited, but competitively, by 5α-androstane-3,17-dione (31) and 5α-androstan-17β-ol-3-one (23) with the same of 25 μM . The activity of S. denitrificans Chol-1ST Δ1-KSTD was strongly and competitively inhibited by corticosterone (46) and estrone (39) with values of about 28 and 68 μM, respectively . Likewise, a Δ1-KSTD from R. rhodochrous IFO 3338 was very sensitive to competitive inhibition by 1-androstene-3,17-dione (17) and estrone (39) with values of 11 and 26.2 μM, respectively . In contrast, a Δ1-KSTD from C. testosteroni ATCC 11996 was less sensitive to estrone . Apparently, inhibition by a steroid is specific to a particular Δ1-KSTD and, thus, as yet it AP20187 is not possible to generalize the inhibition of Δ1-KSTD by steroids.
3-Ketosteroid Δ1-dehydrogenase and 1(2)-hydrogenation activity Although not as widely known as the microbial 1(2)-dehydrogenation of 3-ketosteroids, the reverse reaction, i.e. 1(2)-hydrogenation, has also been reported for several microorganisms. Fermentation of prednisone (49) with Streptomyces hydrogenans suggested 4-pregnene-17α,20β,21-triol-3,11-dione (41) as a possible product , indicating that a 1(2)-hydrogenation had taken place. Likewise, N. simplex and Bacterium cyclo-oxydans were reported to reduce both the C-1,2 double bond and the C-20 ketone of triamcinolone (52) . Such hydrogenation was also reported for baker’s yeast, Saccharomyces cerevisiae . The question is whether the 1(2)-dehydrogenation is catalyzed by a Δ1-KSTD or by another enzyme. In N. simplex VKM Ac-2033D, 1(2)-dehydrogenation and 1(2)-hydrogenation activities were reported to be two separable activities . Similarly, a partially purified steroid 1(2)-hydrogenase from the AD-producing Mycobacterium sp. NRRL B-3805 was apparently different from the other known Δ1-KSTDs and failed to display 1(2)-dehydrogenase activity on 3-ketosteroids . However, by adjusting the medium composition and aeration rate, 1(2)-dehydrogenation and 1(2)-hydrogenation of 3-ketosteroids in N. simplex ATCC 6946 and Bacterium cyclo-oxydans ATCC 12673 [119,120], N. simplex VKM Ac-2033D , as well as in R. erythropolis IMET 7030 and IMET 7185, M. smegmatis IMET SG 99, and M. phlei IMET SG 1026 [122,123] were shown to be reversible and performed by seemingly the same enzyme. An important indication of the in vitro enzymatic 1(2)-hydrogenation of 3-ketosteroids was obtained with a cell-free extract preparation of a Δ1-KSTD from B. sphaericus ATCC 7055. Incubation of ADD (9) with a fraction of the cell-free extract in the presence of 3H2O resulted in a small quantity of highly radioactive AD (8) . Furthermore, a highly purified Δ1-KSTD from R. erythropolis IMET 7030 was demonstrated to act both as a 1(2)-dehydrogenase on AD and as a 1(2)-hydrogenase on ADD in the presence of the electron donor Na2S2O4 . Likewise, a pure Δ1-KSTD from R. rhodochrous IFO 3338 catalyzed 1(2)-hydrogenation of ADD using as electron donor Na2S2O4-reduced benzyl viologen under anaerobic conditions . Having both 1(2)-dehydrogenase and 1(2)-hydrogenase capabilities, the Δ1-KSTD enzymes from R. erythropolis IMET 7030 and R. rhodochrous IFO 3338 were able to catalyze 1(2)-transhydrogenation between 3-keto-4-ene-steroids and 3-keto-1,4-diene-steroids [83,96]. For example, in the presence of ADD, 17α-methyltestosterone (26) was 1(2)-dehydrogenated to 1-dehydro-17α-methyltestosterone (27), while ADD was 1(2)-hydrogenated to AD by the Δ1-KSTD from R. erythropolis IMET 7030 . Using D2O as the 1(2)-transhydrogenation medium, it was shown that the enzymes abstract 1α- and 2β-hydrogen atoms from a 3-keto-4-ene-steroid, transfer the 1α-hydrogen atom to a 3-keto-1,4-diene-steroid and release the 2β-hydrogen atom to the medium. The transhydrogenation was reported to be reversible; initially, the catalytic reaction proceeds rapidly, and, with increasing product concentration, it decreases until equilibrium is reached [83,96]. Kinetic studies suggested that the transhydrogenation proceeds with a typical ping-pong mechanism .