Clostridium orbiscindens sp. nov., a Human Intestinal Bacterium Capable of Cleaving the Flavonoid C-Ring

Winter, J.; Popoff, Michel‐Robert; Grimont, Patrick A. D.; Bokkenheuser, V.

doi:10.1099/00207713-41-3-355

Cited by 110 publications

(88 citation statements)

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“…An early study on flavonoid degradation by a soil pseudomonad speculated on the presence of oxygenases based on the oxidation products and proposed that the degradation proceeded via protocatechuate production (46). The anaerobic degradation of flavonoids by the intestinal microflora, in comparison to the aerobic pathways, has been well documented, and several reports describe the reduction and dehydroxylation reactions leading to phloroglucinol formation (10,15,26,43,44,54,55).This study was undertaken to elucidate the pathway for utilization of the archetypal flavonoid, quercetin, in a plant growth-promoting rhizobacterial (PGPR) strain of Pseudomonas putida which can also grow on naringenin, daidzein, apigenin, hespertin, naringin, protocatechuate, p-hydroxy benzaldehyde, or p-hydroxy benzoic acid as the sole carbon source. In order to understand the catabolism of quercetin in Pseudomonas, we constructed several transposon insertion mutants defective in flavonoid catabolism and compared the metabolic profiles of pathway intermediates in the cultures of wild-type and three mutant strains by using high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy.…”

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“…Efficient utilization of these metabolites can lead to a positive selection of the utilizers. It is, therefore, not surprising that several secondary metabolite degraders have been reported (8,22,23,26,39,41,52,55). Flavonoids belong to a class of 15-carbon secondary metabolites that have a three-ringed structure and are mostly found conjugated with glycosides.…”

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Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Pillai

Swarup

2002

Appl Environ Microbiol

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Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying miniTn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase highpressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.A diverse group of compounds of plant origin are ubiquitously exuded into the rhizosphere by roots. Such compounds are known to affect microbial growth by serving as nutrient sources, through their antimicrobial activities, or by directly affecting microbial gene expression (12,13,16,18,31,34,36,42,51,53). Among the various root exudates, the most diverse classes of signal molecules are the plant secondary metabolites. Efficient utilization of these metabolites can lead to a positive selection of the utilizers. It is, therefore, not surprising that several secondary metabolite degraders have been reported (8,22,23,26,39,41,52,55). Flavonoids belong to a class of 15-carbon secondary metabolites that have a three-ringed structure and are mostly found conjugated with glycosides. Due to their rich carbon content, flavonoids have potential significance as nutritional sources, whereas their complex structures allow them to have diverse pharmacological and physiological effects (30).Several microbes, such as Rhizobia, Agrobacterium, Pseudomonas, Bacillus, and Rhodococcus spp., that exist in the rhizosphere are known to participate in the breakdown or degradation of flavonoids (2, 3). Although, some mechanisms have been proposed, the pathways were not elucidated. A survey of flavonoid-degrading rhizobial strains revealed that flavonoids were generally cleaved via C-ring fission (38, 39). In particular, there is a dearth of information on the biochemical and genetic aspects of catabolism of flavonoids in Pseudomonas spp. An early stud...

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Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Pillai

Swarup

2002

Appl Environ Microbiol

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“…Eubacterium ramulus and Flavonifractor plautii (formerly Clostridium orbiscindens) are human intestinal bacteria that have been demonstrated to cleave the central heterocyclic C-ring of flavonols and flavones (9)(10)(11)(12). Both species are strict anaerobes and common inhabitants of the human intestine (12,13).…”

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Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

et al. 2016

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The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (؉)-taxifolin but not that of (؊)-taxifolin. The K m for (؉)-taxifolin was 6.4 ؎ 0.8 M, and the V max was 108 ؎ 4 mol min ؊1 (mg protein) ؊1 . The enzyme also isomerized (؉)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus. Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439 -1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus. IMPORTANCE Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus.Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria. F lavonoids represent a major group of plant-derived polyphenolic compounds and have been implicated in beneficial effects on human health (1-4). The flavonoids are classified into several subgroups, which include, among others, flavonols, flavanonols, flavones, flavanones, and chalcones. Quercetin (Fig. 1) is a highly abundant dietary flavonoid that shows a broad range of biological activities. Thus, this flavonol has been intensively studied with respect to its role in disease prevention and use in therapy (5-7). The effects of quercetin and other flavonoids depend on how they are metabolized in the human body, including their conversion by intestinal bacteria (8). Beside deglycosylation and deconjugation, intestinal bacteria are also able to further transform the resulting flavonoid aglycones. However, knowledge about the corresponding bacterial species and, in particular, the enzymes involved is still limited.Eubacterium ramulus and Flavonifractor plautii (formerly Clostridium orbiscindens) are human intestinal bacteria that have been demonstrated to cleave the central heterocyc...

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“…Bacteroides thetaiotaomicron is well known for its highly diverse and adaptive metabolic capabilities (9, 40). As for the genus Clostridium and the species Escherichia coli, they have been shown to be able to metabolize other polycyclic compounds such as flavonoids (2,39). In their investigations on the microbial metabolism of IQ, Carman et al have focused their research of bioactive bacteria on individual representatives of the genus Eubacterium belonging to various environments, e.g., blood, mouth, and feces (4).…”

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¹ H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5- f ]Quinoline by Human Intestinal Microbiota

Humblot

Combourieu

Väisänen

et al. 2005

Appl Environ Microbiol

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2-Amino-3-methylimidazo[4,5-f]quinoline (IQ)is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 M) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ 1 H nuclear magnetic resonance ( 1 H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and 1 H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction: Bacteroides thetaiotaomicron (n ‫؍‬ 2), Clostridium clostridiiforme (n ‫؍‬ 3), Clostridium perfringens (n ‫؍‬ 1), and Escherichia coli (n ‫؍‬ 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.

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Clostridium orbiscindens sp. nov., a Human Intestinal Bacterium Capable of Cleaving the Flavonoid C-Ring

Cited by 110 publications

References 11 publications

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

¹ H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5- f ]Quinoline by Human Intestinal Microbiota

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Clostridium orbiscindens sp. nov., a Human Intestinal Bacterium Capable of Cleaving the Flavonoid C-Ring

Cited by 110 publications

References 11 publications

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

1 H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5- f ]Quinoline by Human Intestinal Microbiota

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¹ H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5- f ]Quinoline by Human Intestinal Microbiota