Human Intestinal Anaerobe Research Articles

An NADH-Dependent Reductase from Eubacterium ramulus Catalyzes the Stereospecific Heteroring Cleavage of Flavanones and Flavanonols.

The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (2S,3S)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed.IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.

The ability of human intestinal anaerobes to metabolize different oligosaccharides: Novel means for microbiota modulation?

Prebiotic oligosaccharides are known to have significant impacts on gut microbiota and are thus widely used to program healthy microbiota composition and activity from infants to the elderly. Bifidobacteria and lactobacilli are among the major target microorganisms of oligosaccharides, but the metabolic properties of oligosaccharides in other predominant gut microbes have not been well characterized. In the present study, we demonstrated the metabolic properties of six oligosaccharides in 31 key gut anaerobes. Bifidobacteria readily metabolized fructooligosaccharide (FOSs) with degree of polymerization (DP) 3, i.e. 1-kestose, but several strains used did not actively metabolize FOSs with DP4 and DP5, i.e. nystose and fructosylnystose. Akkermansia muciniphila, a potential new probiotic against obesity, did not show significant growth with any of the oligosaccharides tested. The butyrate producer Anaerostipes caccae grew well on 1-kestose but poorly on FOS mixtures, whereas it contained 1-kestose at 30%. Bacteroides-Parabacteroides group species were separated into two groups based on oligosaccharide metabolic properties. One group metabolized well most of the oligosaccharides tested, but the others metabolized only 1 or 2 selected oligosaccharides. Oligosaccharide profiles after culturing revealed that Bifidobacterium spp. preferentially metabolized shorter oligosaccharides (DP3) in the mixtures, whereas Bacteroides-Parabacteroides spp. did not show oligosaccharide selectivity for metabolism or rather preferred longer oligosaccharides (>DP4). The fermentation profiles indicated specific links between the microbial end-products and specific gut microbes. Available carbohydrates had a significant impact on the accumulation of amino acid-derived bacterial metabolites (i.e. phenol, p-cresol, indole and skatole) and short chain fatty acids. The results assist in predicting the impact of oligosaccharides in human intervention and gut microbiota modulation.

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols.

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 Km for (+)-taxifolin was 6.4 ± 0.8 μM, and the Vmax was 108 ± 4 μmol 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.

Enzymes responsible for the metabolism of saikosaponins from Eubacterium sp. A-44, a human intestinal anaerobe.

From a human intestinal bacterium, Eubacterium sp. A-44, which is capable of hydrolyzing saikosaponins to saikogenins, two glycosidases, beta-D-glucosidase and a novel type of beta-D-fucosidase, were isolated and characterized as saikosaponin-hydrolyzing beta-D-glucosidase and prosaikogenin-hydrolyzing beta-D-fucosidase. Relative to the hydrolyzing activities toward saikosaponins a, b1 and b2, the beta-D-glucosidase showed lower ability to hydrolyze saikosaponin d, but no ability to hydrolyze saikosaponin c or prosaikogenins. By Sephacryl S-300 column chromatography, the molecular weight of prosaikogenin-hydrolyzing beta-D-fucosidase was estimated to be about 130 kDa. The beta-D-fucosidase could hydrolyze prosaikogenins A and F, but not prosaikogenins D and G or saikosaponins. Relative to p-nitrophenyl beta-D-fucoside-hydrolyzing activity, this enzyme had 32.0% and 22.2% of its hydrolyzing ability toward p-nitrophenyl beta-D-glucoside and p-nitrophenyl beta-D-galactoside, respectively. p-Nitrophenyl beta-D-fucoside-hydrolyzing activity was inhibited by D-fucose, and was weakly inhibited by D-glucose, D-glucono delta-lactone, D-galactose and D-galactono delta-lactone. By combining these two glycosidases, saikosaponins a and b1 were converted to their saikogenins via the corresponding prosaikogenins.

Purification and characterization of a novel sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe.

A novel beta-glucosidase, which is inducible and capable of catalyzing the hydrolysis of sennosides, was purified from Bifidobacterium sp. strain SEN with Triton X-100 solubilization and DEAE-cellulose column chromatography, by which hydrolytic activities toward sennoside B, 4-methylumbelliferyl beta-glucoside (MUG), and p-nitrophenyl beta-glucoside (pNPG) were obtained together in the same eluted fractions. The activity was stable against detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, but was denatured by SDS and beta-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polyacrylamide gel electrophoresis (PAGE) either after the enzyme was denatured or when it was not denatured. In the non-denaturing SDS-PAGE, a single protein band hydrolyzed MUG on the gel. In the denaturing SDS-PAGE, the subunit mass of the enzyme was estimated to be 110 kDa. The enzyme was optimally active at pH 6.0 for hydrolysis of sennoside B and MUG. Km values for sennoside B and MUG are 0.94 and 0.53 mM, respectively. The enzyme also catalyzed the hydrolysis of pNPG, amygdalin, geniposide and salicin. It was less active against methyl beta-glucoside and incapable of hydrolyzing cellobiose. The beta-glucosidase activity was inhibited by deoxynojirimycin and p-chloromercuribenzenesulfonic acid, but was less susceptible to several metals (FeSO4, ZnCl2, and CuSO4), and 5,5'-dithio-bis(2-nitrobenzoic acid).

A purgative action of barbaloin is induced by Eubacterium sp. strain BAR, a human intestinal anaerobe, capable of transforming barbaloin to aloe-emodin anthrone.

Orally administered barbaloin (100 mg/kg) did not induce any diarrhea in male Wistar rats, in spite of severe diarrhea with sennoside B (40 mg/kg). Also, in gnotobiote rats mono-associated with Peptostreptococcus intermedius, a human intestinal anaerobe capable of reducing sennidins to rhein anthrone, barbaloin did not induce diarrhea; the faecal water content (71.9%) 8 h after the administration of barbaloin was not increased, compared with that (73.9%) just before the treatment. However, severe diarrhea was induced with barbaloin in gnotobiote rats mono-associated with Eubacterium sp. strain BAR, another human intestinal anaerobe capable of transforming barbaloin to aloe-emodin anthrone; the faecal water content was significantly increased to 85.5% 8 h after the administration, from 73.2% before the treatment. At this time, barbaloin was transformed to aloe-emodin anthrone in the feces from the gnotobiote rats mono-associated with the strain BAR, but not in feces from the conventional rats or the gnotobiote rats mono-associated with P. intermedius. These facts indicate that barbaloin is inactive as a laxative itself but is activated to aloe-emodin anthrone, a genuine purgative component, by Eubacterium sp. strain BAR.

Purification and characterization of a geniposide-hydrolyzing beta-glucosidase from Eubacterium sp. A-44, a strict anaerobe from human feces.

A geniposide-hydrolyzing beta-glucosidase was discovered in Eubacterium sp. A-44, a human intestinal anaerobe. The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography. The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences. A broad substrate specificity was shown for the beta-glucosidase to hydrolyze aryl beta-D-glucosides (p-nitrophenyl beta-D-glucopyranoside-pNPG, esculin and salicin), alkyl beta-D-glucosides (geniposide and amygdalin) and cellobiose. The Km values (mM) for various beta-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose. The pH optimum with pNPG and geniposide as the substrates was 6.0. The enzyme was inhibited by sulfhydryl reagents, Cu2+, and nojirimycin bisulfite.

Enzymic studies on the animal and intestinal bacterial metabolism of geniposide.

Geniposide, a main iridoid glucoside of Gardenia fruit, is transformed to genipin, a genuine choleretic, in vivo in rats (Aburada et al., J. Pharmacobio-Dyn., 1, 81 (1978)). As geniposide was not hydrolyzed to any metabolite by rat liver homogenate, which has beta-D-glucosidase and esterase activities, beta-D-glucosidases in intestinal bacteria seem to be required for an exhibition of its choleretic action. The crude extract of Eubacterium sp. A-44, a human intestinal anaerobe, hydrolyzed geniposide, but that of Ruminococcus sp. PO1-3, another human anaerobe, did not, though both extracts had beta-D-glucosidase activities for p-nitrophenyl beta-D-glucopyranoside. Only one of three beta-D-glucosidases from E. sp. A-44 and none of two from R. sp. PO1-3 hydrolyzed geniposide to genipin. However, carboxylesterases from E. sp. A-44 and pig liver were unable to hydrolyze geniposide to geniposidic acid, but hydrolyzed genipin to an aglycone of geniposidic acid, indicating that geniposide is first hydrolyzed to genipin by beta-D-glucosidases and subsequently to the aglycone of geniposidic acid by esterases. Thus, when geniposide is orally administered, genipin seems to be effectively produced in the intestine and then absorbed to act as a genuine choleretic.

Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans

The dietary carcinogen, 2-amino-3-methyl-3 H-imidazo[4,5-ƒ]quinoline (IQ) is mutagenic in the Salmonella/microsomal mutagenicity assay when activated by microsomal enzymes. IQ is found in many cooked foods, notably fried beef and pork. In laboratory rodents IQ is carcinogenic. We showed that mixed and pure cultures of human intestinal anaerobes, notably Eubacterium spp., metabolized IQ to 2-amino-3,6-dihydro-3-methyl-7 H-imidazo[4,5-ƒ]quinoline-7-one (HOIQ). Unlike IQ, both the synthetic and bacterially produced HOIQ were direct-acting mutagens, i.e. active without microsomal activation. This new direct-acting mutagen, from the bacterial metabolism of a dietary pyrolysis carcinogen, raises new concerns about the possible role of this class of genotoxins in the etiology of human cancer.

Production of a mutagen from ponceau 3R by a human intestinal anaerobe

Ponceau 3R was reduced in vitro by Fusobacterium sp. 2, a human intestinal anaerobe, to a product which is mutagenic when metabolically activated by liver S9 preparations in the Salmonella/mammalian-microsome mutagenicity test. This mutagenic metabolite has been identified as 2,4,5-trimethylaniline.

Azo reduction of trypan blue to a known carcinogen by a cell-free extract of a human intestinal anaerobe

The azo reductase activity of a cell-free extract of Fusobacterium sp. 2 is characterized using trypan blue as a substrate. Either chemical reduction of this dye with sodium hydrosulfite or reduction by the cell-free extract produces a mutagenic product, o-tolidine. The o-tolidine is mutagenic in the Ames Salmonella/mammalian-microsome mutagenicity test when activated by a rat liver S 9 preparation.

A new hydrolase specific for taurine-conjugates of bile acids.

Through the investigation of the bile acid-deconjugation activities of human intestinal anaerobes, a new enzyme was discovered in Peptostreptococcus intermedius which hydrolyzed specifically the taurine-conjugates, but not the glycine-conjugates of bile acids. However, the enzymes in Streptococcus faecalis and Lactobacillus brevis hydrolyzed chiefly the glycine-conjugates.

21-dehydroxylation of corticoids by anaerobic bacteria isolated from human fecal flora.

Two human intestinal anaerobes, culture No. 116 and Eubacterium lentum, synthesize enzymes capable of metabolizing C-21 steroid hormones. 21-Dehydroxylase activity by culture No. 116 benefits from the presence of Escherichia coli or allied organisms in the medium; their effect appears to be a lowering of the Eh. The enzymes activity is independent of arginine and shows no sign of product inhibition. The enzyme, which is constitutive, functions at pH 6.5–7.3; its activity is unrelated to the 4-ene-3CO group but is inhibited by a C20-OH group. E. lentum (neotype), in addition to a 21-dehydroxylase, synthesizes a 3α-hydroxysteroid dehydrogenase active at Eh ± —150 mV, which explains the epimerization of 3α-pregnanolone observed in cultures of fecal flora. The metabolic alterations of deoxycorticosterone by cultures of fecal flora may be duplicated by Clostridium paraputrificum and E. lentum, whether acting in concert or sequentially.