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  • KSTDs are generally reported to be intracellular enzymes eit

    2020-05-14

    — Δ1-KSTDs are generally reported to be intracellular enzymes, either soluble or bound to subcellular particles. For instance, the enzymes from C. testosteroni ATCC 11996 and ATCC 17410 [50,90,99], R. equi [29], and N. simplex ATCC 6946 [52] were particulate-bound. On the other hand, the Δ1-KSTDs from B. sphaericus ATCC 7055 [66], R. rhodochrous IFO 3338 [27], S. denitrificans Chol-1ST [47] and A. fumigatus CICC 40167 [100] were considered to be soluble. However, several bacteria, including N. simplex VKM Ac-2033D (formerly Arthrobacter globiformis 193) [101,102], R. erythropolis IMET 7030 [84,103,104,105,106], and Mycobacterium sp. VKM Ac1817D [107], were shown to produce both soluble and particulate-bound Δ1-KSTDs. This property is likely to be protein-dependent rather than species-dependent, but it may also depend on the particular substrate to be converted, as for instance shown by M. fortuitum ATCC 6842, which produced a cytoplasmic membrane-bound Δ1-KSTD when induced with AD (8), but a soluble isoenzyme when induced with 9α-hydroxyprogesterone (44) [53]. Surprisingly, extracellular Δ1-KSTD activities were found in the fermentation broths of M. neoaurum (formerly Mycobacterium sp. and M. vaccae) VKM Ac-1815D [108] and Mycobacterium sp. VKM Ac1817D [107]. However, the extracellular Δ1-KSTD from M. neoaurum VKM Ac-1815D was associated with a 3β-hydroxysteroid oxidase secreted by the 2 types of active transport [108], which may have triggered the secretion of the Δ1-KSTD. Thus, it appears that Δ1-KSTD activities are localized mostly inside the cell, which makes sense in view of the requirement of reducing the prosthetic group after the reaction. — The experimentally determined molecular masses of Δ1-KSTDs are around 53-61 kDa [27,28,47,48,62,90,93,100,105,109]. The Δ1-KSTDs from R. rhodochrous IFO 3338 [27], N. simplex IFO 12069 [48], and R. erythropolis SQ1 isoenzyme 1 [30] are monomeric proteins, whereas the enzyme from S. denitrificans Chol-1ST [47] forms soluble oligomeric aggregates. Uniquely, the Δ1-KSTD from Mycobacterium sp. VKM Ac-1817D with a molecular mass of ˜58 kDa was proposed to be a dimer consisting of 34 and 23 kDa protein subunits [107]. — Δ1-KSTDs from R. rhodochrous IFO 3338 [27] and R. erythropolis IMET 7030 [84,93,105] were identified as acidic proteins with isoelectric point (pI) values of 3.1 and 4.7, respectively. However, pI calculations using the ProtParam tool (http://web.expasy.org/compute_pi/) suggest that the pIs of (putative) Δ1-KSTDs currently available in GenPept vary considerably, ranging from 4.5 (R. qingshengii BKS 20-40; GenPept EME18626) to 9.6 (Cupriavidus necator; GenPept WP_042876660). — The characterized Δ1-KSTDs generally show optimum activity at basic conditions (pH 8.0-10.0) [27,29,48,49,50,92,110]. Indeed, Δ1-KSTD1 from R. erythropolis SQ1 is most stable at pH 9.0 [111]. Since Δ1-KSTDs employ a catalytic base to abstract a proton from its substrate [30,96,97,98], the basic environment may strengthen the basic character of the catalytic base. In contrast, the enzyme from S. denitrificans Chol-1ST was reported to have its maximum activity at pH 6.0 [47]. Thus, like the pI, the optimum pH for activity of Δ1-KSTDs appears to vary quite significantly among the enzymes.
    AD (8), a central microbial cholesterol degradation intermediate after removal of the C-17 side chain, is a common substrate of Δ1-KSTDs. Almost all characterized Δ1-KSTDs effectively catalyze 1(2)-dehydrogenation of this 3-ketosteroid [27,28,29,47,48,49,50,51,52,53]. In addition, Δ1-KSTDs are also able to 1(2)-dehydrogenate a wide range of other steroid substrates with varying activity. Substituents at the C3 position — The presence of a keto group at the C3 position of the steroid substrates is crucial for catalysis by Δ1-KSTDs. Δ1-KSTD from C. testosteroni ATCC 11996 was found to 1(2)-dehydrogenate various 3-ketosteroids such as 19-nor-4-androstene-3,17-dione (28), cortexolone (47), and progesterone (43), but not 3-hydroxysteroids, including 3α-hydroxy-5α-androstan-17-one (29) and 3β-hydroxy-5α-androstan-17-one (30) [50]. Similar observations have been reported for the Δ1-KSTDs from R. equi [29], N. simplex ATCC 6946 and IFO 12069 [48,49,51,52], Clostridium paraputrificum [92], M. fortuitum ATCC 6842 [53], R. rhodochrous IFO 3338 [27], R. erythropolis SQ1 [28], S. denitrificans Chol-1ST [47], and M. neoaurum ATCC 25795 [62]. The Δ1-KSTD from S. denitrificans Chol-1ST was also inactive on steroids lacking a functional group at the C3 position such as 5α-cholestane (58) [47]. On the basis of these observations the steroid C3 keto group was proposed to interact with an electrophilic or proton donating residue(s) of the enzyme to promote keto-enol tautomerization and labilization of the C2 hydrogen atoms [30,95,96,97,98]. Indeed, in the crystal structure of the Δ1-KSTD1•ADD complex [30], the steroid C3 keto group was observed at hydrogen bonding distance from the hydroxyl group of Tyr-487 and the backbone amide of Gly-491 (Fig. 4), two amino acid residues that are absolutely conserved among Δ1-KSTDs (Supplementary Figure S2).