br Four active site residues of KSTD are fully conserved
Four active site residues of Δ1-KSTD1 are fully conserved in Δ1-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 Δ1-KSTD1•ADD complex revealed that the hydroxyl group of Tyr-318 is at reaction distance to the C2 AZ 10606120 dihydrochloride 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 Δ1-KSTDs. Indeed, mutating them confirmed their catalytic importance , and their roles in catalysis were assigned by analogy with the structure and mechanism of Δ4-(5α)-KSTD , an enzyme with a similar 3D structure to that of Δ1-KSTD1 (see below; ).
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 . Thus, as discussed above, sustained dehydrogenation by Δ1-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 Δ1-KSTD is unknown, although vitamin K2(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 Δ1-KSTD, i.e. 3-ketosteroid 1(2)-dehydrogenation, has been described (see below; ). Dehydration or dehydrogenation? — Enzymatic carbon-carbon double bond formations commonly proceed either via dehydration (e.g. by fumarase) or via dehydrogenation (e.g. by acyl coenzyme A dehydrogenase). In the case of dehydration, the introduction of a double bond into a hydrocarbon moiety, such as at the C1-C2 position of 3-ketosteroids, would require the introduction of a hydroxyl group, which is then followed by a dehydration reaction . However, several observations suggested that this route does not apply to Δ1-KSTDs. For instance, the Δ1-KSTDs are fully functional in anaerobic conditions [27,29,47,50,66], while many enzymatic hydroxylations require molecular oxygen . Moreover, 1α-, 1β-, and 2α-hydroxysteroids are not substrates for Δ1-KSTDs [29,50], and 2α-hydroxytestosterone is 1(2)-dehydrogenated, instead of dehydrated, by B. sphaericus ATCC 7055 Δ1-KSTD . Therefore, analogous to other flavoenzymes, it was already early on postulated that the reaction catalyzed by Δ1-KSTD most likely proceeds via a direct elimination of two adjacent hydrogen atoms from its substrate, i.e. dehydrogenation [29,50]. Which hydrogen atoms are removed? — The Δ1-KSTD-catalyzed dehydrogenation is generally accepted as proceeding via a trans-diaxial elimination with removal of the axial α-hydrogen from the C1 atom and the axial β-hydrogen from the C2 atom of the 3-ketosteroid substrate [30,52,95,96,97,98]. The presence of the α-hydrogen appeared to be an absolute requirement for reactivity. 1α-Substituted 3-ketosteroids were not 1(2)-dehydrogenated by Δ1-KSTDs, but 1β-substituted analogs gave excellent 1(2)-dehydrogenation products under the same conditions [52,95,98]. Accordingly, experiments using deuterium-labeled substrates indicated that all hydrogen atoms released from the C1 atom during 1(2)-dehydrogenation by Δ1-KSTDs originated specifically from the α-position [96,98]. Furthermore, in Δ1-KSTD-catalyzed 1(2)-transhydrogenations, the 1α-hydrogen of a 3-keto-4-ene-steroid was transferred directly to a 3-keto-1,4-diene-steroid that served as the electron acceptor [83,96]. On the other hand, the presence of the 2β-hydrogen is not obligatory. B. sphaericus ATCC 7055 Δ1-KSTD completely 1(2)-dehydrogenated not only 2α-substituted 3-ketosteroids, but also 2β-hydroxy-3-ketosteroids, albeit with a poorer conversion yield [95,98]. Furthermore, the enzyme 1(2)-dehydrogenated 2β-deutero-5α-androstane-3,17-dione (cf. 31) with only 86% rather than 100% depletion of the deuterium [97,98], also indicating that the enzyme is not fully specific for removal of the 2β-hydrogen atom. This lack of specificity could be due to the formation of a transient reactive species that may undergo fast exchange of the C2 hydrogen with solvent [83,96,97,98]. Apart from Δ1-KSTD, the trans-diaxial dehydrogenation mechanism was also observed for Δ4-(5α)-KSTD . Such a mechanism is highly similar to that of acyl coenzyme A dehydrogenases [133,134].