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    • br Enzymes involved in microbial

    2019-08-13


    Enzymes involved in microbial steroid degradation are generally not expressed constitutively, but they are upregulated depending on which steroid substrates are present [17,81]. Thus, a cell-free extract prepared from testosterone-adapted C. testosteroni ATCC 11996 cells displayed a 1(2)-dehydrogenation specific activity that was about 50 times higher than that of a cell-free extract prepared from unadapted cells [50]. In addition, such induction is species specific. Although testosterone (24) was a good Δ1-KSTD inducer for C. testosteroni ATCC 11996, it had a poor effect on R. equi. The best tested inducer for this latter bacterium was progesterone (43), which increased the 1(2)-dehydrogenation specific activity about 8-fold compared to steroid-uninduced cells [29]. Furthermore, the induction is also steroid specific. Particular steroids, e.g. cortisol (48), were 1(2)-dehydrogenated slowly by Septomyxa affinis and, therefore, were termed \"slow\" steroids. Indeed, the dehydrogenation could be accelerated by adding a small quantity of a second steroid as stronger inducer, such as progesterone, AD (8), 3-oxo-23,24-bisnor-4-cholen-22-ol (54), 3-oxo-23,24-bisnor-4-cholen-22-al (55), or 3-oxo-23,24-bisnor-4-cholen-22-oic special health care needs (56) [82]. Similar inductions were also reported for Δ1-KSTD expression in many other microorganisms, such as R. erythropolis (formerly Nocardia erythropolis) IMET 7185 [83], R. erythropolis (formerly Nocardia opaca and R. rhodochrous) IMET 7030 [84], and Bacillus cereus [85]. However, there may also be growth stage differences: for instance, in the spores of F. solani a Δ1-KSTD is expressed constitutively, but in the mycelium state of the fungus it is induced [86].
    Electron acceptor — Removal of cellular debris from Δ1-KSTD-containing cell extracts resulted in the loss of almost all 1(2)-dehydrogenating activity [27,47,66,87]. However, the activity could be restored by adding external electron acceptors such as phenazine methosulfate, menadione, 2,6-dichlorophenol-indophenol, resazurin, Wurster\'s blue, methylene blue, coenzyme Qs, or vitamin Ks [27,29,47,50,66,87,88,89,90,91]. Molecular oxygen has also been reported to act as an external electron acceptor for Δ1-KSTDs from Clostridium paraputrificum [92], R. rhodochrous (formerly Nocardia corallina) IFO 3338 [27], and R. erythropolis SQ1 isoenzyme 3 [28]. On the other hand, a number of other electron acceptors were not compatible, including FAD, FMN, DPN, TPN, NAD+, NADP+, cytochrome c, and coenzyme Q10 [27,29,47,50,66,91]. While ferricyanide was generally reported not to be a good electron acceptor for the activity of Δ1-KSTDs, it was active with the enzyme from the denitrifying Gram-negative bacterium S. denitrificans Chol-1ST [47]. The nature and role of the prosthetic group — As mentioned above, Δ1-KSTDs can utilize either phenazine methosulfate or 2,6-dichlorophenol-indophenol as the external electron acceptor. Moreover, the enzyme is strongly inhibited by acriflavin [29,50]. Since these properties have also been observed for various flavoproteins, it was proposed already early on that Δ1-KSTDs might use flavin as a prosthetic group for their dehydrogenating activity [29,50]. This hypothesis was supported by the bright yellow colour of purified Δ1-KSTDs that exhibited absorption maxima around 270, 370, and 460 nm, which are typical for flavoproteins [27,30,47,48,93]. Final proof of the nature of the prosthetic group was obtained from reconstitution experiments with purified apo-Δ1-KSTD. Only when FAD was added to the apo-enzyme, the activity was fully restored, thus identifying FAD as the prosthetic group of Δ1-KSTD [27,94]. Crystal structures of R. erythropolis SQ1 Δ1-KSTD1 showed that one FAD is bound per enzyme molecule through non-covalent interactions only, including hydrogen bonds, van der Waals contacts, and dipole-dipole interactions [30]. Nevertheless, the binding is tight, with a dissociation constant of 0.075 for the Δ1-KSTD from R. erythropolis IMET 7030 [94], and 4.7 μM for the Δ1-KSTD from R. rhodochrous IFO 3338 [27]. The role of the prosthetic group during steroid 1(2)-dehydrogenation is essential; presumably it accepts the axial α-hydrogen (see Fig. 4) from the C1 atom of the steroid substrate as a hydride ion [95,96,97,98]. Indeed, this hypothesis was confirmed by the crystal structure of the Δ1-KSTD1•ADD complex, in which the N5 atom of the isoalloxazine ring of the FAD prosthetic group is positioned at the α-side of ADD, at reaction distance to the C1 atom of the steroid, suitable to accept a hydride ion from the C1 atom [30].