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  • inno-206 kinase Since these first results KSTD activity has

    2019-09-24

    — Since these first results, Δ1-KSTD activity has been identified in many other microorganisms, albeit sometimes with different substrate preferences. For instance, Comamonas testosteroni ATCC 11996 (formerly Pseudomonas testosteroni) is active on several steroid substrates, but it cannot use 11β-hydroxy and 11-keto steroids such as inno-206 kinase (48) and cortisone (53), because its Δ1-KSTD is not active toward these substrates [50]. Similarly, R. equi can completely degrade progesterone (43), but degradation of A-nor-testosterone (21) halts at 9α-hydroxy-A-nor-4-androstene-3,17-dione (33), since its Δ1-KSTD cannot oxidize the 5-membered A ring of this substrate [57]. Evidence for the essentiality of Δ1-KSTD comes from Δ1-KSTD-defective bacterial strains, such as M. fortuitum NRRL B-8119 [58], M. roseum sp. nov. 1108/1 [59], and Mycobacterium sp. VKM Ac1817D [46]. These strains degrade their steroid substrates only up to the Δ1-KSTD substrate 9-OHAD (34). Likewise, inactivation of tesH, the Δ1-KSTD gene in C. testosteroni TA441, destroyed its capability to grow on testosterone and resulted in accumulation of AD (8) and 9-OHAD [26]. More recently, it was shown that disruption of the Δ1-KSTD gene of M. tuberculosis H37Rv gave rise to growth attenuation and 9-OHAD accumulation with cholesterol as sole carbon source [60,61]. Finally, the importance of Δ1-KSTD in microbial steroid degradation is also reflected by the frequent presence of multiple Δ1-KSTD genes in steroid-degrading microorganisms. Inactivation of two out of three Δ1-KSTD genes in R. erythropolis SQ1 still allowed the resulting mutant to grow on cholesterol without accumulation of any steroid intermediates [28]. On the other hand, disruption of all identified Δ1-KSTD genes in M. neoaurum ATCC 25795 resulted in a mutant that is still able to degrade inno-206 kinase cholesterol, but only up to 9-OHAD [62]. Interestingly, while R. ruber Chol-4 harbors three genes for Δ1-KSTDs, i.e. kstD1, kstD2, and kstD3, a double-gene deletion of kstD2 and kstD3 was sufficient to completely abolish its capability to grow in minimal medium with cholesterol (1) as the only carbon source [63]. Together, these observations strongly support that Δ1-KSTDs are essential enzymes for microbial steroid degradation. Sequence of early steps in steroid ring opening under aerobic conditions — Depending on the organism, the 1(2)-dehydrogenation and 9α-hydroxylation of AD (8) to yield the unstable intermediate 9-OHADD (10) can occur sequentially, i.e. 1(2)-dehydrogenation followed by 9α-hydroxylation or the other way round, or simultaneously. In the incomplete ring-A aromatization of AD with a species of Pseudomonas studied by Dodson and Muir [42] ADD (9) was one of the products, implying that the bacterium first 1(2)-dehydrogenates AD to ADD and subsequently hydroxylates ADD at C-9 to 9-OHADD. The same sequence of events was suggested for the conversion of AD with R. ruber strain Chol-4, as ADD was detected as main intermediate in the course of the fermentation [63]. Likewise, M. tuberculosis H37Rv most likely uses the same route to open the steroid B-ring, since its 3-ketosteroid 9α-hydroxylase enzyme displayed a clear preference for ADD over AD [31]. On the other hand, the opposite sequence was suggested for aromatization-degradation of AD with a species of Nocardia A20-10. As stated above, from a fermentation of AD using this bacterium, 9-OHAD was isolated from the mixture with 3-HSA (11), indicating that 9α-hydroxylation followed by 1(2)-dehydrogenation took place [43]. Furthermore, with R. erythropolis SQ1 1(2)-dehydrogenation and 9α-hydroxylation were proposed to occur simultaneously in the conversion of AD to 9-OHADD with a preference for 9α-hydroxylation followed by 1(2)-dehydrogenation to keep a low intracellular ADD concentration [64]. Two Δ1-KSTD isoenzymes of strain SQ1 involved in this conversion showed comparable affinities (KM values) for AD and 9-OHAD [28], but a high ADD concentration was moderately toxic to the bacterium [64,65]. Thus, a microbial species may use one of the above-mentioned three available routes to convert AD to 9-OHADD. However, the possibility of the species to switch from one route to another, depending on which 3-ketosteroid(s) are available, may apply as well.