The role and mechanism of microbial 3-ketosteroid Δ1-dehydrogenases in steroid breakdown

3-Ketosteroid Δ1-dehydrogenases are FAD-dependent enzymes that catalyze the introduction of a double bond between the C1 and C2 atoms of the A-ring of 3-ketosteroid substrates. These enzymes are found in a large variety of microorganisms, especially in bacteria belonging to the phylum Actinobacteria. They play a critical role in the early steps of the degradation of the steroid core. 3-Ketosteroid Δ1-dehydrogenases are of particular interest for the etiology of some infectious diseases, for the production of starting materials for the pharmaceutical industry, and for environmental bioremediation applications. Here we summarize and discuss the biochemical and enzymological properties of these enzymes, their microbial sources, and their natural diversity. The three-dimensional structure of a 3-ketosteroid Δ1-dehydrogenase in connection with the enzyme mechanism is highlighted.


Introduction
Sterols are an abundant source of steroids in nature and a large variety of microorganisms are able to transform them, either partially, or completely to carbon dioxide and water. One such sterol is cholesterol (1 in Fig. 1). Its complex chemical structure requires the concerted action of a large number of enzymes to completely degrade it. The occurrence of genes coding for cholesterol-degrading enzymes in several bacterial and fungal genome sequences [1], indicates that cholesterol degradation pathways may be active in a variety of microorganisms.

Importance of Δ 1 -KSTDs
Since these first results, Δ 1 -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 cortisol (48) and cortisone (53), because its Δ 1 -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-Anor-4-androstene-3,17-dione (33), since its Δ 1 -KSTD cannot oxidize the 5-membered A ring of this substrate [57]. Evidence for the essentiality of Δ 1 -KSTD comes from Δ 1 -KSTD-defective bacterial strains, such as M. fortuitum NRRL B-8119 [58], M. roseum sp. nov. 1108/1 [59], and Mycobacterium sp. VKM Ac1817D [107]. These strains degrade their steroid substrates only up to the Δ 1 -KSTD substrate 9-OHAD (34). Likewise, inactivation of tesH, the Δ 1 -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 Δ 1 -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 Δ 1 -KSTD in microbial steroid degradation is also reflected by the frequent presence of multiple Δ 1 -KSTD genes in steroid-degrading microorganisms. Inactivation of two out of three Δ 1 -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 Δ 1 -KSTD genes in M. neoaurum ATCC 25795 resulted in a mutant that is still able to degrade cholesterol, but only up to 9-OHAD [62]. Interestingly, while R. ruber Chol-4 harbors three genes for Δ 1 -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 Δ 1 -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 9αhydroxylation followed by 1(2)-dehydrogenation took place [43]. Furthermore, with R. erythropolis SQ1 1(2)-dehydrogenation and 9αhydroxylation 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 Δ 1 -KSTD isoenzymes of strain SQ1 involved in this conversion showed comparable affinities (K M 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 abovementioned 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.
A phylogenetic analysis of Δ 1 -KSTD sequences resulted in a cladogram with several different clades, i.e. clades A, B, C, and D (Fig. 3). All Δ 1 -KSTDs from fungi of the phylum Ascomycota and bacteria of the phylum Chloroflexi are clustered in subclade A1, which also includes an archaeal Δ 1 -KSTD from Candidatus Caldiarchaeum subterraneum. Subclade A2 contains actinobacterial Δ 1 -KSTDs in one cluster and proteobacterial enzymes in the other cluster. Subclade B1 mostly contains Δ 1 -KSTDs from Firmicutes bacteria and the amoebozoa P. pallidum PN500. Subclade B2 is mostly occupied by actinobacterial enzymes, but it also comprises a Δ 1 -KSTD from the bacterium Empedobacter falsenii, a member of the phylum Bacteroidetes. Although Δ 1 -KSTDs from Actinobacteria can be found in virtually all clades, the majority of these enzymes are in subclade B2 and in clade C. Similarly, the enzymes from Proteobacteria are present in several clades, but mainly clustered in clade D. This latter clade also accommodates some actinobacterial Δ 1 -KSTDs. Hence, in general, Δ 1 -KSTDs are phylogenetically clustered on the basis of their microbial sources.

Distribution of isoenzymes in the phylogenetic tree
In the cladogram, multiple Δ 1 -KSTD isoenzymes of a particular organism tend to be distributed across several clades, instead of clustered in a single clade. For instance, the five Δ 1 -KSTD isoenzymes from the actinobacterium R. opacus PD630 appear in clades B (subclades B1 and B2), C, and D. Similarly, the Δ 1 -KSTD isoenzymes from the actinobacteria R. erythropolis, N. simplex, and M. neoaurum, as well as the proteobacterium Novosphingobium malaysiense are found in several different clades. If the presence in different clades is correlated with differences in substrate specificity, as suggested above, these distributions may reflect the capability of the corresponding microorganisms to use a diverse variety of steroid substrates.

Steroid inducibility of Δ 1 -KSTD
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 Δ 1 -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) [82]. Similar inductions were also reported for Δ 1 -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 Δ 1 -KSTD is expressed constitutively, but in the mycelium state of the fungus it is induced [86].

The nature and role of the prosthetic group
As mentioned above, Δ 1 -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 Δ 1 -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 Δ 1 -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-Δ 1 -KSTD. Only when FAD was added to the apo-enzyme, the activity was fully restored, thus identifying FAD as the prosthetic group of Δ 1 -KSTD [27,94]. Crystal structures of R. erythropolis SQ1 Δ 1 -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 μM for the Δ 1 -KSTD from R. erythropolis IMET 7030 [94], and 4.7 μM for the Δ 1 -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 Δ 1 -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].

Cellular location of Δ 1 -KSTDs
Δ 1 -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] Fig. 3. Unrooted phylogenetic analysis of Δ 1 -KSTDs. Δ 1 -KSTD protein sequences were obtained from the NCBI protein database using all variants of the enzyme name as queries. To prepare a non-redundant size-reduced dataset, the sequences with more than 60% identity were clustered using the program CD-HIT [78]. The cluster-representing sequences were aligned to the amino acid sequence of the structurally characterized Δ 1 -KSTD1 from Rhodococcus erythropolis SQ1 [30] using ClustalW2 [79] and visually inspected for the details of their alignment; the sequences that are incomplete and/or do not conserve the key amino acid residues, i.e. the residues that correspond to Tyr-119, Tyr-318, Tyr-487, and Gly-491 of Δ 1 -KSTD1, were removed from the dataset. All biochemically characterized Δ 1 -KSTD sequences (*) were then included into the dataset. Likewise, multiple Δ 1 -KSTD isoenzyme sequences from several species (bold) were also added. The sequences in the resulting dataset were multiplyaligned with ClustalW2 and the cladogram was obtained from this alignment using the Neighbor-Joining method [80] implemented in the program MEGA6 [75]. The taxa identifications are: phylum|species-the NCBI protein data-base accession number; the phyla Act, Asc, Bac, Chl, Fir, Myc, Pro, and Tha stand for Actinobacteria, Ascomycota, Bacteroidetes, Chloroflexi, Firmicutes, Mycetozoa, Proteobacteria, and Thaumarchaeota, respectively. and Mycobacterium sp. VKM Ac1817D [107], were shown to produce both soluble and particulate-bound Δ 1 -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 Δ 1 -KSTD when induced with AD (8), but a soluble isoenzyme when induced with 9α-hydroxyprogesterone (44) [53]. Surprisingly, extracellular Δ 1 -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 Δ 1 -KSTD from M. neoaurum VKM Ac-1815D was associated with a 3β-hydroxysteroid oxidase secreted by the cells [108], which may have triggered the secretion of the Δ 1 -KSTD.
Thus, it appears that Δ 1 -KSTD activities are localized mostly inside the cell, which makes sense in view of the requirement of reducing the prosthetic group after the reaction.
An important indication of the in vitro enzymatic 1(2)-hydrogenation of 3-ketosteroids was obtained with a cell-free extract preparation of a Δ 1 -KSTD from B. sphaericus ATCC 7055. Incubation of ADD (9) with a fraction of the cell-free extract in the presence of 3 H 2 O resulted in a small quantity of highly radioactive AD (8) [66]. Furthermore, a highly purified Δ 1 -KSTD from R. erythropolis IMET 7030 was demonstrated to act both as a 1(2)-dehydrogenase on AD and as a 1(2)-hydrogenase on ADD in the presence of the electron donor Na 2 S 2 O 4 [124]. Likewise, a pure Δ 1 -KSTD from R. rhodochrous IFO 3338 catalyzed 1(2)-hydrogenation of ADD using as electron donor Na 2 S 2 O 4 -reduced benzyl viologen under anaerobic conditions [96].

Overall fold
High-resolution crystal structures of Δ 1 -KSTD are currently available for the Δ 1 -KSTD1 isoenzyme from R. erythropolis SQ1 [30]. The Δ 1 -KSTD1 molecule has an elongated shape, and consists of two domains, an FAD-binding domain and a catalytic domain, which are connected by a two-stranded antiparallel β-sheet. The FAD-binding domain adopts a Rossmann fold, a characteristic nucleotide-binding fold, with a basic topology of a symmetrical α/β structure composed of two halves of β1-α1-β2-α2-β3 and β4-α4-β5-α5-β6 connected at the β3 and β4 strands by an α-helix (α3) crossover [125,126]. However, some minor modifications to the basic topology were observed in the FAD-binding domain, in which the third β-strand of the second half is missing and the α-helix crossover is replaced by a three-stranded β-meander. The catalytic domain contains a four-stranded antiparallel β-sheet surrounded by several α-helices and a small double-stranded antiparallel β-sheet [30].
In Δ 1 -KSTD1, the FAD adopts an extended conformation with an almost planar isoalloxazine ring system, similar to what has been found in proteins belonging to the glutathione reductase family [125]. It fits in an elongated cavity in the FAD-binding domain. Its adenine end is in front of the parallel β-sheet of the Rossmann fold, while its isoalloxazine ring is at the interface of the FAD-binding and catalytic domains. The si-face of the isoalloxazine ring (see Fig. 4) interacts with the FAD-binding domain, while the re-face is oriented towards the catalytic domain, and the O4, C4A, N5, and C5A atoms face the bulk solvent [30].

Active site
Δ 1 -KSTD1 possesses a pocket-like active site cavity that is suitable for binding a steroid ring system. It is located at the interface between the FAD-binding and the catalytic domains, near the FAD-binding site. The active site is lined with hydrophobic amino acid residues originating from both domains and bordered by the re-face of the isoalloxazine ring of the FAD prosthetic group [30]. The hydrophobic nature of the residues that line the active site is conserved among Δ 1 -KSTD enzymes (Supplementary Figure S2).
The structure of the Δ 1 -KSTD1•ADD complex showed that 3-ketosteroids are bound by the enzyme via a large number of van der Waals interactions, a hydrophobic stacking interaction, and two hydrogen bonds to the C3 carbonyl oxygen atom via the Tyr-487 hydroxyl group and the Gly-491 backbone amide. The A-ring of the 3-ketosteroid aligns almost parallel to the plane of the isoalloxazine ring. It is deeply buried in the active site and sandwiched between the re-face of the pyrimidine moiety of the isoalloxazine ring on its α-side and residues Tyr-119 and Tyr-318 on its β-side. This arrangement places the C1 and C2 atoms of the 3-ketosteroid at short distances to the N5 atom of the isoalloxazine ring and the Tyr-318 hydroxyl group, respectively. On the other hand, the five-membered D-ring of the 3-ketosteroid occupies a solvent-accessible pocket near the active site entrance [30].
As evidenced by the NCBI protein database, Δ 1 -KSTD sequences have been identified in a large number of microbial species. However, their amino acid sequences are rather similar to the Δ 1 -KSTD1 sequence (Supplementary Figure S2). The sequence that was most divergent from Δ 1 -KSTD1, was that of a Δ 1 -KSTD from the Gram-negative bacterium Achromobacter xylosoxidans (GenPept CKI19020.1), with an identity of 33%. Homology modeling with this latter sequence on the basis of the Δ 1 -KSTD1 structure, using the Swiss-Model server [129], produced a model that showed that the substrate-binding and the FAD-binding residues are highly conserved. Thus, it can be expected that the majority of the currently identified Δ 1 -KSTDs share a similar overall fold with Δ 1 -KSTD1.

Key residues of Δ 1 -KSTD
Four active site residues of Δ 1 -KSTD1 are fully conserved in Δ 1 -KSTDs from different species (Supplementary Figure S2). These residues are Tyr-119, Tyr-487, and Gly-491 from the FAD-binding domain and Tyr-318 from the catalytic domain. The structure of the Δ 1 -KSTD1•ADD complex revealed that the hydroxyl group of Tyr-318 is at reaction distance to the C2 atom of the 3-ketosteroid ligand, while the hydroxyl group of Tyr-487 and the backbone amide of Gly-491 make hydrogen bonds with the C3 carbonyl oxygen atom. Although Tyr-119 has no close contacts with the bound ADD in the complex structure, its hydroxyl group is at hydrogen-bonding distance to the hydroxyl group of Tyr-318. Their absolute conservation and their interaction with ADD suggested that the residues are important for activity of Δ 1 -KSTDs. Indeed, mutating them confirmed their catalytic importance [30], and their roles in catalysis were assigned by analogy with the structure and mechanism of Δ 4 -(5α)-KSTD [127], an enzyme with a similar 3D structure to that of Δ 1 -KSTD1 (see below; [30]).

Catalytic mechanism of Δ 1 -KSTD
A complete catalytic cycle of a flavoenzyme always involves two half-reactions, i.e. a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction the flavin prosthetic group is reduced by the substrate, whereas in the oxidative half-reaction the reduced prosthetic group is re-oxidized by an electron acceptor [130]. Thus, as discussed above, sustained dehydrogenation by Δ 1 -KSTDs is only possible in the presence of an electron acceptor [27,29,47,50,66,[87][88][89][90][91]. At present, the physiological electron acceptor of the oxidative half-reaction of Δ 1 -KSTD is unknown, although vitamin K 2 (35) [88,89] and molecular oxygen [28,92,102] have been proposed as possible electron acceptors. Clearly, the details of electron transfer still need further investigation. On the other hand, a detailed catalytic mechanism of the reductive half-reaction of Δ 1 -KSTD, i.e. 3-ketosteroid 1(2)-dehydrogenation, has been described (see below; [30]).

Are the hydrogens removed simultaneously or one by one?
Δ 1 -KSTDs can catalyze the exchange of alkali-labile tritium or deuterium atoms at the C2 atom of their substrates, even when enzyme turnover was prevented by the absence of an electron acceptor for the oxidative half-reaction [97,98] or by keeping the flavin prosthetic group in the reduced state [96]. This observation indicates that the enzymes more likely employ a stepwise unimolecular elimination conjugate base (E1cB) mechanism, in which departure of the first hydrogen atom precedes that of the second hydrogen atom. Such a mechanism requires the formation of an intermediate. A concerted bimolecular elimination (E2) mechanism, in which the two hydrogens depart simultaneously without the formation of an intermediate, is less likely. Thus, 1(2)-dehydrogenation by Δ 1 -KSTD has been considered to involve a two-step mechanism, i.e. an initial fast step followed by a slow ratedetermining step [98]. The fast step was proposed to be initiated by an interaction of the C3 carbonyl group of the 3-ketosteroid substrate with an electrophile. This interaction stimulates labilization of the C2 hydrogen atoms. Subsequent abstraction of a proton from this atom by a general base results in either an enolate [97,98] or a carbanionic [96] intermediate. In the slow step, a double bond is proposed to be formed between the C1 and C2 atoms when a hydride ion is transferred from the C1 atom of the intermediate to the flavin prosthetic group [96][97][98]. This proposed step-wise mechanism is in contrast to the concerted removal of the hydrogens catalyzed by acyl coenzyme A dehydrogenases [133,134].

Information from the crystal structure of Δ 1 -KSTD
The nature and positions of the amino-acid residues involved in catalysis by Δ 1 -KSTD were clarified with the structure determination of the Δ 1 -KSTD1•ADD complex combined with mutational studies on the enzyme. A superposition of the structure of the substrate AD (8) on that of the product ADD (9) as bound in the Δ 1 -KSTD1•ADD complex structure revealed that 1) the hydroxyl group of Tyr-487 and the backbone amide of Gly-491 would be at the right positions for hydrogen bond formation with the C3 carbonyl group of the substrate; 2) the hydroxyl group of Tyr-318 would be at˜3.0 Å from the C2 atom of the substrate; 3) the N5 atom of the FAD isoalloxazine ring would be at˜2.6 Å from the C1 atom of the substrate; and 4) the isoalloxazine ring and Tyr-318 would be on opposite sides of the A-ring of the substrate, with the isoalloxazine ring at the α-side and Tyr-318 at the β-side. Thus, the substrate would be bound in the active site such that its C1 and C2 atoms are positioned appropriately for hydride and proton abstraction, respectively [30].
These observations facilitated a detailed description of the 1(2)dehydrogenation mechanism of Δ 1 -KSTD1 (Fig. 4) [30]. Tyr-487 and Gly-491 tightly bind the carbonyl oxygen of the 3-ketosteroid substrate to promote keto-enol tautomerization and labilization of the C2 hydrogen atoms. The hydroxyl group of Tyr-318 serves as a general base that abstracts the axial β-hydrogen from the C2 atom as a proton. A transient carbanionic intermediate, which is most likely stabilized by keto-enol tautomerization, is formed. This negatively charged intermediate can be stabilized by the delocalization of its charge over the C3 keto group and the interaction with the positive N-terminal helix macro-dipole of a nearby α-helix. Tyr-119, whose hydroxyl group is hydrogen bonded to the Tyr-318 hydroxyl group, may increase the basic character of Tyr-318 and facilitate proton relay to the solvent. The negative charge of the intermediate is then shifted to the C1 atom to form the double bond between the C1 and C2 atoms. In synchrony, the N5 atom of the FAD prosthetic group abstracts the axial α-hydrogen from the C1 atom as a hydride ion, generating a reduced anionic FAD. The negative charge of this anion can be delocalized over the pyrimidine moiety of the isoalloxazine prosthetic group. The pyrimidine moiety is stabilized by hydrogen bonding interactions with the protein backbone as well as by the helix macro-dipole interaction with the Nterminal end of an α-helix. Re-oxidation of the reduced FAD by an electron acceptor in the subsequent oxidative half-reaction will complete the catalytic cycle and make the enzyme available for another cycle [30].

Concluding remarks
A crucial step in the microbial degradation of steroids is the 1(2)dehydrogenation of the steroid nucleus by FAD-dependent Δ 1 -KSTDs. This step is required to initiate the opening of the steroid nucleus under both aerobic and anaerobic conditions. A large variety of steroid-degrading microorganisms can carry out this biotransformation or harbor gene(s) encoding (putative) Δ 1 -KSTD(s), attesting the enzyme's physiological importance and role. Many microorganisms, particularly from the phylum Actinobacteria, may even express multiple Δ 1 -KSTD isoenzymes. In line with their widespread distribution, Δ 1 -KSTDs have quite diverse amino acid sequences. Yet, even the most deviating sequences appear to be compatible with the R. erythropolis SQ1 Δ 1 -KSTD1 fold, suggesting that Δ 1 -KSTDs share a common overall fold. Biochemical, structural, and mutational studies substantiated that Δ 1 -KSTDs catalyze a direct 1(2)-dehydrogenation of 3-ketosteroid substrates. The enzymes make use of a Tyr residue to abstract the axial βhydrogen from the C2 atom of the substrate as a proton and use FAD to accept the axial α-hydrogen from the C1 atom as a hydride ion. To complete the catalytic cycle, the reduced FAD should be re-oxidized by an electron acceptor. However, the nature of the electron acceptor and mechanism of the re-oxidation are currently incompletely understood and need further investigation. Finally, although AD is a common substrate for Δ 1 -KSTDs, most biochemically-characterized enzymes are able to accept a wide range of naturally occurring and chemically modified 3-ketosteroids as substrates. How the Δ 1 -KSTDs fine-tune their substrate specificity is an intriguing subject for further investigation, which may be of interest for future biotechnological development and production of specialty steroids.