Bacteria on steroids: the enzymatic mechanism of an NADH-dependent dehydrogenase that regulates the conversion of cortisol to androgen in the gut microbiome

Abstract

The ability to metabolize both endogenous and exogenous compounds to a variety of metabolic products is not exclusive to our human cells. In fact, the bacterial communities that inhabit our digestive system are responsible for a network of steroid transformations that can produce hormones in the gut, which are then absorbed to act in the host. These communities have been shown to impact our health in numerous ways, affecting disease predisposition, pathogenesis, physical fitness, and dietary responsiveness. Steroid biotransformations by gut bacteria are predicted to impact the host endocrine system. A particular set of transformations facilitated by microbial enzymes has been shown to result in the formation of 11-oxy-androgens from hostderived cortisol. Since androgens have been implicated in disease and immune modulations, understanding the structure and catalytic mechanism of enzymes involved in cortisol metabolism is a key step to hasten the development of strategies that reduce the formation of disease-promoting bioactive steroids in certain individuals. Here, we combine experimental and computational techniques to describe DesC, an enzyme capable of creating 20a-dihydrocortisol and siphoning cortisol away from pathways that produce androgens. DesC diverges significantly from previously described bacterial and eukaryotic counterparts, catalyzing an NADH-dependent 20ahydroxysteroid dehydrogenase reaction but presenting little sequence and structure similarity to them. The structural information obtained by X-ray crystallography and hybrid QM/MM simulations, validated through mutagenesis studies, show the reaction occurs through a multi-step proton relay mechanism. Free energy calculations were then used to describe the kinetics of the reaction mechanism. The mechanistic information presented here can be employed in the development of therapeutics to divert microbial pathways away from disease-promoting steroids. AUTHOR INFORMATION Corresponding Author

Microbial Hydroxysteroid Dehydrogenases: From Alpha to Omega

“…Recently, the C. scindens ATCC 35704 20α-HSDH was crystallized for further characterization of the enzymatic mechanism. Hybrid quantum mechanical molecular modeling simulations revealed a reaction mechanism involving a multistep proton relay, which was validated by site-directed mutagenesis experiments of active site and substrate binding residues [16]. An amino-acid homology search based on C. scindens ATCC 35704 20α-HSDH within the National Center for Biotechnology Information (NCBI) database uncovered two additional organisms, Denitratisoma oestradiolicum DSM 16959 and Intestinibacillus sp.…”

“…Hydroxysteroid dehydrogenases belong to one of the following three large and diverse protein superfamilies: short-chain dehydrogenase/reductase (SDR), medium-chain dehydrogenase/reductase (MDR), or aldo-keto reductase (AKR) [5,14]. Many SDR and MDR family hydroxysteroid dehydrogenases have been identified in the gut microbiome [14][15][16][17]. HSDHs in the AKR superfamily are generally found within mammals [12], although microbial AKR family HSDHs have been reported [18].…”

“…The zinc-containing MDRs share a strictly conserved Gly, His, and Glu for zinc binding [27]. MDR family HSDHs include host BA 3β-HSD [26] and microbial glucocorticoid 20α-HSDH [14,16].…”

Completion of the gut microbial epi-bile acid pathway

“…To date, only one steroid 20α-HSDH sequence, which interconverts cortisol and 20α-dihydrocortisol, has been reported ( C. scindens EDS07887.1). 44 , 45 Therefore, we performed a BLASTP search and found two sequences with high similarity, WP_145772308.1 from Denitratisoma oestradiolicum DSM 16959 and WP_107631222.1 from Intestinibacillus sp. Marseille-P4005.…”

Completion of the gut microbial epi-bile acid pathway

“…13, 15, 41 7α- and 7β-HSDH were shown in numerous Firmicutes 14, 37, 65 and the Actinobacteria Collinsella aerofaciens . 25 Along with these bile acid-specific HSDHs, the glucocorticoid 20α- and 20β-HSDHs are evident in both Firmicutes 43, 45 and Actinobacteria such as Bifidobacterium adolescentis 66 . Until this study, there were no reports of genes encoding 12β-HSDH and the activity had only been shown in C. paraputrificum , C. tertium and C. difficile.…”