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  • br Structure of ketosteroid dehydrogenase Overall fold High

    2020-03-21


    Structure of 3-ketosteroid Δ1-dehydrogenase Overall fold — High-resolution crystal structures of Δ1-KSTD are currently available for the Δ1-KSTD1 isoenzyme from R. erythropolis SQ1 [30]. The Δ1-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]. The structure of Δ1-KSTD1 is most similar to that of a 3-ketosteroid Δ4-(5α)-dehydrogenase (Δ4-(5α)-KSTD) from R. jostii RHA1 (PDB 4at0 [127]; 28% sequence identity). The next similar structure is a flavocytochrome c fumarate reductase from Shewanella putrefaciens MR-1 (PDB 1d4c [128]; 24% sequence identity). This is not very surprising because Δ1-KSTD1 and the two other proteins are all FAD-dependent enzymes with very similar functions; Δ1-KSTD1 1(2)-dehydrogenates 3-ketosteroids [30] with a possibility to be reversible (see below), Δ4-(5α)-KSTD 4(5)-dehydrogenates 3-keto-(5α)-steroids [127], while the fumarate reductase hydrogenates (reduces) a carbon-carbon double bond of fumarate [128]. 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 IFN-alpha 1, human recombinant protein 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 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 IFN-alpha 1, human recombinant protein 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.