TY - JOUR
T1 - 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
AU - Mythen, Sean M.
AU - Devendran, Saravanan
AU - Méndez-García, Celia
AU - Cann, Isaac
AU - Ridlon, Jason M.
N1 - Publisher Copyright:
© 2018 American Society for Microbiology.
PY - 2018/4/1
Y1 - 2018/4/1
N2 - 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 HSDHencoding genes difficult and necessitating biochemical characterization. We located a gene cluster in Eggerthella sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in Escherichia coli. 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.
AB - 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 HSDHencoding genes difficult and necessitating biochemical characterization. We located a gene cluster in Eggerthella sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in Escherichia coli. 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.
KW - Bile acid
KW - Eggerthella
KW - Hydroxysteroid dehydrogenase
KW - Metagenome
KW - Targeted gene synthesis
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U2 - 10.1128/AEM.02475-17
DO - 10.1128/AEM.02475-17
M3 - Article
C2 - 29330189
AN - SCOPUS:85044105720
SN - 0099-2240
VL - 84
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
IS - 7
M1 - e02475-17
ER -