In KSTD the FAD adopts an extended

In Δ1-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 — Δ1-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 Bromfenac Sodium 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 Δ1-KSTD enzymes (Supplementary Figure S2). The structure of the Δ1-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, Δ1-KSTD sequences have been identified in a large number of microbial species. However, their amino acid sequences are rather similar to the Δ1-KSTD1 sequence (Supplementary Figure S2). The sequence that was most divergent from Δ1-KSTD1, was that of a Δ1-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 Δ1-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 Δ1-KSTDs share a similar overall fold with Δ1-KSTD1.
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 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 Δ1-KSTDs. Indeed, mutating them confirmed their catalytic importance [30], and their roles in catalysis were assigned by analogy with the structure and mechanism of Δ4-(5α)-KSTD [127], an enzyme with a similar 3D structure to that of Δ1-KSTD1 (see below; [30]).