Biology subjects

Horinouchi, M.

Publications and source records attributed to Horinouchi, M..

8 recordsLinked to original sources

bioRxiv2026

Complete genome sequence of Comamonas testosteroni TA441 and comparative analysis of steroid degradation gene clusters across Proteobacteria and Actinomycetota

Comamonas testosteroni TA441 is a model aerobic steroid-degrading bacterium whose sterane degradation pathway has been elucidated in greater detail than that of any other bacterium to date. Similar pathways have been identified in many bacterial genera, including members of Proteobacteria and Actinomycetota, such as Mycobacterium tuberculosis. Steroid degradation genes in TA441 form an approximately 120-kb mega-cluster, and 40 genes involved mainly in the degradation of the sterane structure and the C20?C22 portion of the side chain at C17 have been identified. Here, we present the complete genome sequence of TA441 together with our findings on its steroid degradation genes. We also compare the genes in the mega-cluster with corresponding genes in other Comamonas strains, ?-Proteobacteria other than Comamonas, ?-Proteobacteria, ?-Proteobacteria, and Actinomycetota using CAGECAT to analyze similarities and differences in steroid degradation genes among these bacteria.

microbiology↗

bioRxiv2025

Identification of the C9-Hydrogenase for 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid (9,17-DOHNA) and the 7α-Dehydratase Essential for Initiating β-Oxidation of the B-, C-, and D-Rings in Steroid Degradation by Comamonas testosteroni TA441

Comamonas testosteroni TA441 is a model aerobic steroid-degrading bacterium whose sterane degradation pathway has been elucidated in the greatest detail to date. Similar pathways have been identified in many genera of bacteria, including both proteobacteria and actinobacteria such as Mycobacterium tuberculosis. However, the genes encoding the C9-hydrogenase for 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (9,17-DOHNA, also known as HIP) and the 7-dehydratase essential for initiating {beta}-oxidation of the B-, C-, and D-rings had not been identified within the gene cluster responsible for B-, C-, and D-ring degradation. In this study, we identified these missing genes, located adjacent to the chsE1E2H1H2ltp2 cluster involved in C17 side-chain degradation, and designated them scdB and scdH, respectively. This finding completes the elucidation of all degradation steps of 9,17-DOHNA prior to D-ring cleavage. AlphaFold-predicted models showed that the hydrogenases/dehydrogenases involved in steroid degradation in TA441--ScdG, ScdE, 3{beta}-dehydrogenase (3{beta}-DH), SteA, 3-dehydrogenase (3-DH), and SteB--share a similar Rossmann-like /{beta}/ sandwich fold with ScdB and function as dimers. In contrast, ScdH was predicted to form a homohexameric structure, similar to ScdY and ScdN, members of the crotonase-like enoyl-CoA hydratase/isomerase family involved in B-, C-, and D-ring degradation. Furthermore, AlphaFold modeling revealed that SteC, the dehydratase responsible for removing the C12{beta}-hydroxyl group from 9,17-DOHNA derivatives, exhibits strong structural similarity to BaiE, the bile acid 7-dehydratase of Clostridium scindens JCM 10418/VPI 12708, despite sharing only [~]28% amino acid sequence identity.

microbiology↗

bioRxiv2025

Identification of Dehydrogenase, Hydratase, and Aldolase Required for C17 Propionyl Residue Removal in Steroid Degradation by Comamonas testosteroni TA441: Structural and Functional Insights into the (ChsE1-ChsE2)2 and (ChsH1-ChsH2MaoC)2-(Ltp2-ChsH2DUF35)2 Complexes

Bacterial steroid degradation is gaining attention for its diverse roles, such as Mycobacterium tuberculosiss reliance on the degradation of the C17 side chain of cholesterol for survival in host environments. ORF40 to ORF44 of Comamonas testosteroni TA441, previously characterized for its A-, B-, C-, and D-ring cleavage pathways, were hypothesized to correspond to the igr operon of M. tuberculosis, encoding the dehydrogenase ChsE1E2, hydratase ChsH1H2 maoC, and aldolase Ltp2 ChsH2DUF35, responsible for propionyl residue removal in C17 side-chain degradation. However, low amino acid identity between the corresponding enzymes precluded functional assignment without experimental evidence. In this study, we generated gene-disrupted mutants of ORF40-44 and demonstrated that ORF41/44, ORF40/42, and ORF43 encode the dehydrogenase, hydratase, and aldolase, respectively. ORF40 encodes a bifunctional protein comprising MaoC and DUF35 domains. The MaoC domain of ORF40 and the ORF42-encoded protein form the hydratase, while the DUF35 domain is essential for aldolase activity. A mutant expressing ORF40MaoC and ORF40DUF35 separately exhibited both hydratase and aldolase activities, suggesting these activities do not require a strictly formed complex of hydratase and aldolase. However, efficient propionyl residue removal appears to depend on the proper formation of each enzymatic complex, including ChsE1E2, ChsH1H2MaoC, and Ltp2ChsH2DUF35; although (ChsE1-ChsE2)2 does not form a stable complex with (ChsH1-ChsH2MaoC)2-(Ltp2-ChsH2DUF35)2, some degree of interaction was suggested. AlphaFold-predicted 3D structures of the TA441 enzymes and their complexes revealed striking similarities to those of M. tuberculosis, despite low amino acid identities. These findings shed light on the structural and functional conservation of bacterial steroid-degrading enzymes. IMPORTANCEResearch on bacterial steroid degradation began over 50 years ago, primarily to produce substrates for steroid drugs. Recently, the role of steroid-degrading bacteria in human health has garnered increasing attention. Comamonas testosteroni TA441 is a prominent model organism for studying aerobic steroid degradation, with its overall pathways for A-, B-, C-, and D-ring cleavage already elucidated. In this study, we identified the mechanism for removing the propionyl residue at C17, a crucial step in degrading steroids with a C17 side chain, such as cholic acid, cholesterol, and other biologically significant compounds in animals and plants. The functions and structures of the identified enzymes show remarkable similarity to those in Mycobacterium tuberculosis. These findings suggest that insights gained from TA441 could provide valuable clues for understanding M. tuberculosis steroid metabolism and the broader ecological and health-related implications of bacterial steroid degradation.

microbiology↗

bioRxiv2023

Identification of "missing links" in C- and D-ring cleavage of steroids by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization and cleavage of the A- and B-ring, followed by D- and C-ring cleavage via b-oxidation. We previously characterized most of the above degradation steps; however, a few intermediate compounds remained unaccounted for. We hypothesized that cleavage of the D-ring at C13-17 required the ScdY hydratase and was followed by cleavage of the C-ring via the ScdL1L2 transferase. The reaction was expected to produce 6-methyl-3,7-dioxo-decane-1,10-dioic acid-Coenzyme A (CoA) ester. To verify this hypothesis, we constructed a plasmid that enabled targeted gene induction in TA441 mutant strains. The major product of ScdL1L2 was found to be 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester; whereas the substrate of ScdY was revealed to be a geminal diol, 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester. This finding suggests that ScdY catalyzes the addition of a water molecule at C14 of 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester, leading to D-ring cleavage at C13-17. The C9 ketone of the D-ring cleavage product is then converted to a hydroxyl group, followed by C-ring cleavage to produce 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester. Precise bacterial bile acid degradation pathway will be one of the key to investigate the gut-microbiota-brain axis, which affects human health and disease. IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain substrates for steroid drugs. The role of steroid-degrading bacteria in relation to human health is attracting growing attention. Comamonas testosteroni TA441 is the leading bacterial model of aerobic steroid degradation and the overall pathway has been outlined previously. However, a few intermediate compounds in C- and D-ring cleavage processes have remained unknown. Here, we identified the missing compounds and can now propose the complete A-, B-, C-, and D-ring cleavage pathway employed by steroid-degrading bacteria. This finding will facilitate the application of such microorganisms for the synthesis of specific steroid derivatives.

microbiology↗

bioRxiv2023

Comprehensive summary of steroid metabolism in Comamonas testosteroni TA441; entire degradation process of basic four rings and removal of C12 hydroxyl group

Comamonas testosteroni is one of the representative aerobic steroid-degrading bacterium. We previously revealed the mechanism of steroidal A,B,C,D-ring degradation by C. testosteroni TA441. The corresponding genes are located in two clusters at both ends of a mega-cluster of steroid degradation genes. ORF7 and ORF6 are the only two genes in these clusters, whose function has not been determined. Here, we characterized ORF7 as encoding the dehydrase responsible for converting the C12{beta} hydroxyl group to the C10(12) double bond on the C-ring (SteC), and ORF6 as encoding the hydrogenase responsible for converting the C10(12) double bond to a single bond (SteD). SteA and SteB, encoded just upstream of SteC and SteD, are in charge of oxidizing the C12 hydroxyl group to a ketone group, and of reducing the latter to the C12{beta} hydroxyl group, respectively. Therefore, the C12 hydroxyl group in steroids is removed with SteABCD via the C12 ketone and C12{beta} hydroxyl groups. Given the functional characterization of ORF6 and ORF7, we disclose the entire pathway of steroidal A,B,C,D-ring breakdown by C. testosteroni TA441. IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago, primarily to obtain materials for steroid drugs. Now, their implications for the environment and humans, especially in relation to the infection and the brain-gut-microbiota axis, are attracting increasing attention. Comamonas testosteroni TA441 is the leading model of bacterial aerobic steroid degradation with the ability to break down cholic acid, the main component of bile acids. Bile acids are known for their variety of physiological activities according as their substituent group(s). In this study, we identified and functionally characterized the genes for removal of C12 hydroxyl groups and provide a comprehensive summary of the entire A,B,C,D-ring degradation pathway by C. testosteroni TA441 as the representable bacterial aerobic degradation process of the steroid core structure.

microbiology↗

bioRxiv2021

Identification of the CoA-ester intermediates and genes involved in the cleavage and degradation of the steroidal C-ring by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization of the A-ring accompanied by B-ring cleavage, followed by D- and C-ring cleavage. We previously revealed major enzymes and intermediate compounds in A,B-ring cleavage, {beta}-oxidation cycle of the cleaved B-ring, and partial C,D-ring cleavage process. Here, we elucidated the C-ring cleavage and the {beta}-oxidation cycle that follows. ScdL1L2, a 3-ketoacid Coenzyme A (CoA) transferase which belongs to the SugarP_isomerase superfamily, was thought to cleave the C-ring of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA ester, the key intermediate compound in the degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (3a-H-4 [3'-propionic acid]-7a{beta}-methylhexahydro-1,5-indanedione; HIP)-CoA ester in the previous study; however, this study suggested that ScdL1L2 is the isomerase of the derivative with a hydroxyl group at C-14 which cleaves C ring. The subsequent ring-cleaved product was indicated to be converted to 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester mainly by ORF33-encoded CoA-transferase (named ScdJ), followed by dehydrogenation by ORF21 and 22-encoded acyl-CoA dehydrogenase (named ScdM1M2). Then a water molecule is added by ScdN for further degradation by {beta}-oxidation. ScdN is considered to catalyze the last reaction in C,D-ring degradation by the enzymes encoded in the steroid degradation gene cluster tesB to tesR. IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in human is attracting attention. The overall degradation of steroidal four rings is proposed, however there are still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in C. testosteroni, which is one of the most studied representative steroid degrading bacteria and is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption.

microbiology↗

bioRxiv2021

Identification of essential β-oxidation genes and corresponding metabolites for estrogen degradation by actinobacteria

Steroidal estrogens (C18) are contaminants receiving increasing attention due to their endocrine-disrupting activities at sub-nanomolar concentrations. Although estrogens can be eliminated through photodegradation, microbial function is critical for removing estrogens from ecosystems devoid of sunlight exposure including activated sludge, soils, and aquatic sediments. Actinobacteria were found to be key estrogen degraders in manure-contaminated soils and estuarine sediments. Previously, we used the actinobacterium Rhodococcus sp. strain B50 as a model microorganism to identify two oxygenase genes, aedA and aedB, involved in the activation and subsequent cleavage of the estrogenic A-ring, respectively. However, genes responsible for the downstream degradation of estrogen A/B-rings remained completely unknown. In this study, we employed tiered comparative transcriptomics, gene disruption experiments, and mass spectrometry-based metabolite profile analysis to identify estrogen catabolic genes. We observed the up-regulation of thiolase-encoding aedF and aedK in the transcriptome of strain B50 grown with estrone. Consistently, two downstream estrogenic metabolites, 5-oxo-4-norestrogenic acid (C17) and 2,3,4-trinorestrogenic acid (C15), were accumulated in aedF- and aedK-disrupted strain B50 cultures. Disruption of fadD3 [3a-H-4(3-propanoate)-7a{beta}-methylhexahydro-1,5-indanedione (HIP)-coenzyme A ligase gene] in strain B50 resulted in apparent HIP accumulation in estrone-fed cultures, indicating the essential role of fadD3 in actinobacterial estrogen degradation. In addition, we detected a unique meta-cleavage product, 4,5-seco-estrogenic acid (C18), during actinobacterial estrogen degradation. Differentiating the estrogenic metabolite profile and degradation genes of actinobacteria and proteobacteria enables the cost-effective and time-saving identification of potential estrogen degraders in various ecosystems through liquid chromatography-mass spectrometry analysis and polymerase chain reaction-based functional assays.

microbiology↗

bioRxiv2020

Genetic and metabolite biomarkers reveal actinobacteria-mediated estrogen biodegradation in urban estuarine sediment

Steroidal estrogens are often accumulated in urban estuarine sediments worldwide at microgram per gram levels. These aromatic steroids have been classified as endocrine disruptors with an EC50 at sub-nanomolar concentrations and classified as Group 1 carcinogens by the World Health Organization. Microbial degradation is a naturally occurring mechanism that mineralizes estrogens in the biosphere; however, the corresponding genes in estrogen-degrading actinobacteria remain unidentified. In this study, we identified a gene cluster encoding several putative estrogen-degrading genes in actinobacterium Rhodococcus sp. strain B50. Among them, the oecB and oecC genes involved in estrogenic A-ring cleavage were identified through gene-disruption experiments. We also detected the accumulation of two extracellular estrogenic metabolites, including pyridinestrone acid (PEA) and 3a-H-4(3-propanoate)-7a{beta}-methylhexahydro-1,5-indanedione (HIP), in the estrone-fed strain B50 cultures. Since actinobacterial oecC and proteobacterial oecC shared less than 40% sequence identity, oecC could serve as a specific biomarker to differentiate the contribution of actinobacteria and proteobacteria in environmental estrogen degradation. Therefore, oecC and the extracellular metabolites PEA and HIP were used as biomarkers to investigate estrogen biodegradation in an urban estuarine sediment. Interestingly, our data suggested that actinobacteria, rather than alpha-proteobacteria function in sewage treatment plants, are actively degrading estrogens in the urban estuarine sediment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/329094v1_ufig1.gif" ALT="Figure 1"> View larger version (93K): org.highwire.dtl.DTLVardef@17e1e23org.highwire.dtl.DTLVardef@ecc60borg.highwire.dtl.DTLVardef@be03d4org.highwire.dtl.DTLVardef@ed81d6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIsolation of an estrogen-degrading actinobacterium Rhodococcus sp. strain B50 and establishment of a strain B50 genetic manipulation system. C_LIO_LIStrain B50 exhibits a two-fold estrogen degradation rate of that of estrogen-degrading alpha-proteobacteria under the same cultivation conditions. C_LIO_LIFunctional characterization of two oxygenase genes, oecB and oecC, involved in estrogenic A-ring cleavage in actinobacteria. C_LIO_LIIdentification of two extracellular estrogenic metabolites, PEA and HIP, in the estrone-fed strain B50 cultures. C_LIO_LIDetection of actinobacterial oecC sequences as well as PEA and HIP in the estrone-spiked urban estuarine sediments. C_LI

microbiology↗