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  • Substituents at the C position An oxygen containing

    2020-03-24

    Substituents at the C11 position — An oxygen-containing substituent at the C11 position also affects the catalytic activity of Δ1-KSTDs. The Δ1-KSTDs from the Gram-positive bacteria R. equi [29], N. simplex ATCC 6946 and IFO 12069 [48,49,52], and R. rhodochrous IFO 3338 [27] were able to 1(2)-dehydrogenate 11α-hydroxy-, 11β-hydroxy-, or 11-keto-3-ketosteroids, e.g. 11α-hydroxyprogesterone (45), 11β-hydroxy-4-ansdrostene-3,17-dione (36), adrenosterone (37), cortisol (48), cortisone (53) or corticosterone (46). Likewise, Δ1-KSTD1 and Δ1-KSTD2 from R. erythropolis SQ1 were active on cortisol [28]. On the other hand, Δ1-KSTDs from the Gram-negative bacteria C. testosteroni ATCC 11996 [50] and S. denitrificans Chol-1ST [47] were inactive on both 11β-hydroxy- and 11-keto-3-ketosteroids, although the Δ1-KSTD from C. testosteroni ATCC 11996 could 1(2)-dehydrogenate 11α-hydroxytestosterone (25) [50]. In general, 3-ketosteroids oxygenated at C11 are less easily converted by Δ1-KSTDs than their non-oxygenated analogs [27,29,50]. Yet, the Δ1-KSTD from R. equi catalyzed the 1(2)-dehydrogenation of 11α-hydroxy-progesterone (45) at a similar rate as progesterone (43) [29] and cortisone (53) was a good substrate for the Δ1-KSTD from R. rhodochrous IFO 3338 [27]. Thus, an oxygen-containing substituent at the C11 position of 3-ketosteroids may adversely affect conversion by Δ1-KSTDs. Particularly, 11β-hydroxy and 11-keto groups have a negative effect on the activity of the Δ1-KSTDs from Gram-negative bacteria [48]. Substituents at the C17 position — Δ1-KSTDs accept 3-ketosteroid substrates with varying substituents at the C17 position. In the crystal structure of Δ1-KSTD1•ADD, the C17 atom of ADD (9) is exposed to solvent. In agreement with this observation, most characterized Δ1-KSTDs reacted well on 3-ketosteroids with their C17 carbon atom substituted with hydroxyl (e.g. testosterone; 24), ketone (e.g. AD; 8), acyl (e.g. progesterone; 43), or hydroxyacyl groups (e.g. cortexolone; 47) [27,28,29,47,48,49,50,52,53]. Δ1-KSTDs from R. equi [91], N. simplex ATCC 6946 [52], and S. denitrificans Chol-1ST [47] were even able to 1(2)-dehydrogenate 4-cholesten-3-one (2), and a Δ1-KSTD from Clostridium paraputrificum was active on 3-oxo-5β-cholan-24-oic endothelin receptor (57) [92]. Furthermore, a study on M. tuberculosis H37Rv [56] implied that its Δ1-KSTD is active on a steroid with a C17 side chain degradation intermediate. Effects of such substituents on the reactivity of Δ1-KSTDs toward 3-ketosteroids are variable. For instance, testosterone was the best tested substrate for the Δ1-KSTDs from R. equi [29] and N. simplex IFO 12069 [48], but was a somewhat worse substrate for the enzymes from C. testosteroni ATCC 11996 [50] and S. denitrificans Chol-1ST [47]. Furthermore, the activity of the Δ1-KSTD from C. testosteroni ATCC 11996 [50] and the Δ1-KSTD2 from R. erythropolis SQ1 [28] on progesterone was, respectively, about 60 and 185% of their activity on AD. Thus, Δ1-KSTDs can generally accept 3-ketosteroid substrates with diverse substituents at the C17 position, but with varied, not yet understood, effects on their activity.
    Many dehydrogenases, including alcohol dehydrogenase, isocitrate dehydrogenase, and β-hydroxysteroid dehydrogenase, can be inhibited by their own substrate, particularly at elevated substrate concentrations [112]. This substrate inhibition phenomenon was also observed for the Δ1-KSTDs from C. testosteroni ATCC 11996 [50] and R. equi [29] when tested with AD (8) and testosterone (24), respectively, at concentrations over 0.1 mM (Supplementary Table S3). Furthermore, high concentrations of AD, cortisol (48), cortisone (53), cortexolone (47) and progesterone (43) inhibited the activity of N. simplex ATCC 6946 Δ1-KSTD, with maximal activity at approximately 1 and 0.13 mM cortisol and cortisone, respectively [49,52]. Δ1-KSTD may not only be inhibited by their substrate, but also by their product. The degree of product inhibition appears to be variable, however. 1(2)-Dehydrogenation of cortisone, cortexolone, progesterone, 19-nor-testosterone (22), and AD by N. simplex ATCC 6946 Δ1-KSTD was, to some extent, inhibited by their corresponding 1-dehydro analogs, although the conversion of cortisol was not inhibited [49]. Furthermore, kinetic data on the 1(2)-dehydrogenation of 11β,21-dihydroxy-4,17(20)-pregnadiene-3-one (61) and cortisol using Septomyxa affinis Δ1-KSTD could only be explained by including product inhibition [113]. Moreover, the rate of 1(2)-dehydrogenation of AD with a Δ1-KSTD from C. testosteroni ATCC 11996 was slightly reduced in the presence of its product ADD (9) [50], but no inhibition was observed with a Δ1-KSTD from B. sphaericus ATCC 7055 at the concentration tested [97].