DMPO Dehydrogenation by a KSTD is also a crucial step during
— 1(2)-Dehydrogenation by a Δ1-KSTD is also a crucial step during DMPO degradation of the steroid core. Several Δ1-KSTDs were shown to be active under either aerobic or anaerobic conditions [27,29,47,50,66]. Furthermore, the last common intermediate of both aerobic and anaerobic steroid degradation pathways appeared to be the product of Δ1-KSTD activity [14,39,40], i.e. a Δ1-3-ketosteroid. In an anaerobic environment, the C1-C2 double bond of a Δ1-3-ketosteroid is hydrated to result in the corresponding C1-hydroxylated intermediate, which then follows the 2,3-seco pathway to degrade its steroid core (blue arrows in Fig. 1) [14,39,40,41]. Altogether, Δ1-KSTD is essential for microbial steroid degradation. It is required for opening the steroid nucleus under both aerobic and anaerobic conditions.
Microbial sources of 3-ketosteroid Δ1-dehydrogenase — Microbial steroid 1(2)-dehydrogenation was first reported for the fungi Fusarium solani and F. caucasicum, which converted Δ4-pregnene-3,20-diones, AD (8), and Δ5-3β-hydroxy steroids into ADD (9) . Similar transformations were demonstrated for the bacterium Streptomyces lavendulae and the fungus Cylindrocarpon radicicola ATCC 11011, which fermented progesterone (43) into ADD and 1-dehydrotestololactone (40), respectively . Since then, such biotransformations on various steroid substrates have been reported for a large number of fungi and bacteria. Some recent examples of such microorganisms are M. neoaurum DSM 1381 , R. ruber Chol-4 , and Gordonia neofelifaecis NRRL B-59395 . Indeed, a search in the NCBI protein database revealed that putative Δ1-KSTD enzymes are present in almost 500 different microbial species. The large number and variety of microorganisms that may express this enzyme attest to its physiological role and importance. — It has been found that several microorganisms are able to express multiple Δ1-KSTD isoenzymes (Supplementary Table S1). M. fortuitum ATCC 6842 was reported to produce two different Δ1-KSTDs, depending on the steroid inducers applied. When induced with AD (8) a membrane-associated Δ1-KSTD, which was more active toward AD than toward 9-OHAD (34), was upregulated. In contrast, when induced with 9α-hydroxyprogesterone (44) the bacterium expressed a soluble Δ1-KSTD with a higher activity on 9-OHAD than on AD . In R. erythropolis SQ1, three Δ1-KSTD isoenzymes have been found, i.e. Δ1-KSTD1 , Δ1-KSTD2 [65,73], and Δ1-KSTD3 , with different substrate specificities. While Δ1-KSTD1 and Δ1-KSTD2 displayed a broad 3-ketosteroid substrate range with the best activity on 9-OHAD and AD, respectively, Δ1-KSTD3 had a high preference for 5α-androstane-3,17-dione (31) and 5α-testosterone (23) . In M. smegmatis mc2155 two genes, ksdD1 and ksdD2, encode Δ1-KSTD enzymes. Targeted disruption of ksdD1 partially inactivated the cholesterol degradation pathway by this bacterium, leading to the accumulation of the intermediate AD. On the other hand, inactivation of ksdD2 did not affect the degradation pathway. Nevertheless, the enzyme expressed by this latter gene did exhibit Δ1-KSTD activity, albeit low, during mycobacterial growth on cholesterol . Similarly, R. ruber Chol-4 contains three gene copies for Δ1-KSTD, i.e. kstD1, kstD2, and kstD3. While the role of KstD1 remains unclear, the enzymes encoded by kstD2 and kstD3 were verified to be involved in cholesterol utilization by the bacterium. Specifically, KstD2 was important for 1(2)-dehydrogenation of AD and 9-OHAD . More recently, M. neoaurum ATCC 25795 was found to express three Δ1-KSTD isoenzymes, i.e. MN-KstD1 (GenPept ACV13200.1), MN-KstD2 (GenPept AHG53938.1), and MN-KstD3 (GenPept AHG53939.1), with distinct transcriptional responses to steroids. The isoenzymes were able to 1(2)-dehydrogenate AD, 9-OHAD and testosterone (24) but with some significant differences in their substrate preferences. In particular, MN-KstD1 and MN-KstD2 were also active toward 5α-testosterone (23) . The NCBI protein database contains many other species with two or more putative Δ1-KSTD sequences, such as the actinobacterium R. opacus PD630 (GenPepts AHK28217.1, AHK29640.1, AHK29660.1, AHK33894.1, AHK34331.1) and the fungus Aspergillus fumigatus Af293 (GenPepts XP_751348 and XP_753296). Thus, it appears that steroid-degrading microorganisms may use multiple Δ1-KSTDs to 1(2)-dehydrogenate steroids, most probably as a strategy to increase their capability in degrading various steroid substrates.