Targeted Synthesis and Characterization of a Gene Cluster Encoding NAD(P)H-Dependent 3α-, 3β-, and 12α-Hydroxysteroid Dehydrogenases from Eggerthella CAG:298, a Gut Metagenomic Sequence

Abstract

Gut metagenomic sequences provide a rich source of microbial genes, the majority of which are annotated by homology or unknown. Genes and gene pathways that encode enzymes catalyzing biotransformation of host bile acids are important to identify in gut metagenomic sequences due to the importance of bile acids in gut microbiome structure and host physiology. Hydroxysteroid dehydrogenases (HSDHs) are pyridine nucleotide-dependent enzymes with stereospecificity and regiospecificity for bile acid and steroid hydroxyl groups. HSDHs have been identified in several protein families, including medium-chain and short-chain dehydrogenase/reductase families as well as the aldo-keto reductase family. These protein families are large and contain diverse functionalities, making prediction of HSDH-encoding genes difficult and necessitating biochemical characterization. We located a gene cluster in sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in We then screened bile acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-HSDH, and we determined that CDD59474 (3α-HSDH) and CDD59473 (3β-HSDH) constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these HSDHs with other gut bacterial HSDHs and closest homologues in the database revealed predictable clustering of HSDHs by function and identified several likely HSDH sequences from bacteria isolated or sequenced from diverse mammalian and avian gut samples. Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids, with implications for increased/decreased toxicity for gut bacteria and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved in glucocorticoid metabolism and may alter signaling through nuclear receptors such as farnesoid X receptor and G-protein-coupled receptor TGR5. Biochemical or similar approaches are required to fill in many gaps in our ability to link a particular enzymatic function with a nucleic acid or amino acid sequence. In this regard, we have identified a novel 12α-HSDH and a novel set of genes encoding an iso-bile acid pathway (3α-HSDH and 3β-HSDH) involved in epimerization and detoxification of harmful secondary bile acids.

Metabolism of hydrogen gases and bile acids in the gut microbiome

“…Gut microbes are known to be capable of oxidation and reduction in the hydroxyl groups on the 3-, 7-, and 12-carbons of bile acids (Fig. Amino acid sequence analysis suggests that most bacterial HSDHs in the gut microbiota belong to the short-chain alcohol/polyol dehydrogenase family [56][57][58]. The epimerization of bile acid hydroxyl groups (a ↔ b) requires two position-specific bile acid aand b-HSDHs, which generate a stable oxo-bile acid intermediate i.e., 7a-hydroxy ⟷ 7-oxo ⟷ 7bhydroxy.…”

“…3). Bile acid HSDHs have been detected and characterized in numerous genera inhabiting the lumen of the colon, including; Bacteroides, Clostridium, Escherichia, Eggerthella, Eubacterium, Peptostreptococcus, and Ruminococcus [47, [56][57][58]. Bacterial bile acid HSDHs differ in their pH optima, pyridine nucleotide specificity (NAD(H), NADP(H), or both), subunit molecular weight, and gene regulation [47].…”

“…Genes encoding the iso-bile acid pathway 3a-HSDH and 3b-HSDH have recently been reported in strains of E. lenta [57,58]. This led two independent labs to propose classification schemes for E. lenta isolates based on the number and type of hydroxysteroid dehydrogenase activities observed.…”

“…We recently reported genes encoding NADPHdependent 12a-HSDH in E. lenta DSM 2243 and from a metagenomic sequence, Eggerthella CAG:298 [58] Fig. 3.…”

“…Bacterial bile acid HSDHs differ in their pH optima, pyridine nucleotide specificity (NAD(H), NADP(H), or both), subunit molecular weight, and gene regulation [47]. Amino acid sequence analysis suggests that most bacterial HSDHs in the gut microbiota belong to the short-chain alcohol/polyol dehydrogenase family [56][57][58]. Bile acid HSDHs have been detected and characterized in numerous genera inhabiting the lumen of the colon, including; Bacteroides, Clostridium, Escherichia, Eggerthella, Eubacterium, Peptostreptococcus, and Ruminococcus [47,[56][57][58].…”