Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+

Researchers have long studied enzymes that inactivate steroid hormones and contribute to carcinogenesis. The aldo-keto reductase (AKR) superfamily includes enzymes like 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), which plays a role in both steroid metabolism and PAH carcinogenesis. Prior research has shown that 3 alpha-HSD is the only HSD in the AKR family with a known structure. This gap motivated the investigation of its structural features. No prior work had resolved how NADP+ binds in this enzyme or how the active site accommodates substrates. The lack of structural data on similar enzymes in the AKR family also limited understanding of their catalytic mechanisms. The absence of a clear model for substrate orientation in 3 alpha-HSD left uncertainty about how it interacts with steroids. This uncertainty drove the need for a high-resolution structure to clarify the enzyme's function. Understanding the enzyme's structure could help explain its role in both metabolic and carcinogenic processes.

The aim of this study was to determine the three-dimensional structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase in complex with NADP+. The researchers wanted to clarify how this enzyme inactivates steroid hormones and contributes to PAH carcinogenesis. They focused on the enzyme's structure because it is the only HSD in the AKR superfamily with a known structure. The study aimed to identify how NADP+ binds and how the active site accommodates substrates. The researchers also wanted to explore the possibility of a conserved catalytic mechanism across enzyme superfamilies. They hypothesized that the structure might reveal a similar mechanism to the SDR superfamily. The study sought to provide insights into the enzyme's function by analyzing its crystal structure. The researchers proposed that this structure could serve as a model for other AKR enzymes.

The researchers used X-ray crystallography to determine the structure of 3 alpha-HSD in complex with NADP+. They crystallized the enzyme and collected diffraction data at 2.7 Å resolution. The structure was refined to a crystallographic R-factor of 23.4%. The model included the enzyme's alpha/beta barrel and the NADP+ cofactor. The researchers analyzed the binding site at the C-terminal end of the barrel. They compared the structure to other binary complexes in the AKR superfamily. The study also examined the role of Tyr 55 and Lys 84 in catalysis. The researchers used crystal packing contacts to infer substrate-binding interactions.

The structure of 3 alpha-HSD in complex with NADP+ was resolved at 2.7 Å and refined to 23.4% R-factor. NADP+ binds at the C-terminal end of an alpha/beta barrel, similar to other AKR enzymes. However, NADP+ is found in two alternate conformations, likely due to the absence of a salt-linked safety belt. Tyr 55 is positioned as the general acid in the catalytic mechanism, with Lys 84 lowering its pKa. The study supports a proposed mechanism for carbonyl reduction in AKR enzymes. A crystal packing contact involves Trp 227 from a neighboring molecule inserting into an apolar cleft. This cleft contains residues like Leu 54, Trp 86, and Phe 128, suggesting it is the substrate-binding site. A water molecule in the active site may represent the hydroxyl oxygen of a steroid substrate.

The structure of 3 alpha-HSD with NADP+ supports a catalytic mechanism involving Tyr 55 and Lys 84. The absence of a salt-linked safety belt allows NADP+ to adopt two conformations. The apolar cleft near the active site is likely the substrate-binding site. The crystal packing contact with Trp 227 mimics steroid binding and offers insight into substrate orientation. The water molecule in the active site may indicate the position of the hydroxyl oxygen in a steroid. The study suggests that the alpha face of a bound steroid is oriented toward Trp 86. The structure provides a model for other AKR enzymes in the superfamily. The findings may help explain how 3 alpha-HSD contributes to both steroid inactivation and PAH carcinogenesis.