Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

Abstract

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...

Difference in post-stress recovery of the gut microbiome and its altered metabolism after chronic adolescent stress in rats

“…Based on a thorough literature review, some of our results and observations are completely new findings, while several other findings can be supported by previous reports in the literature. For example, previous studies have revealed that Eubacterium in phyla Firmicutes is generally considered a health-promoting gut microbe 41,42 , and this bacterial genus was detected at relatively decreased abundance in the S-Str compared to other groups, which echos the fact that stress generates adverse health effects to the gut microbes. It is also interesting to note that the www.nature.com/scientificreports www.nature.com/scientificreports/ lactate level of rats' cecum samples significantly elevated immediately after the stress event in this study, which could contribute to gut dysbiosis and increased host gut permeability and inflammation 43 .…”

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

“…By combining ERED with CHI, Gall et al observed the conversion of naringenin to phloretin; coexpression of ERED and CHI led to transformation of eriodictyol and homoeriodictyol in addition to naringenin (12). Based on this report, we intended to use ERED as an auxiliary enzyme in order to compare the rate of taxifolin conversion by CHI (14) to that of naringenin conversion. Unexpectedly, it turned out that phloretin formation from naringenin depended on ERED but not CHI concentration and occurred even in the absence of CHI.…”

“…The conversion of flavanonols by enzymes from E. ramulus depends on the stereoisomeric form and the availability of NADH. While (2R,3R)-stereoisomers are isomerized by CHI to the corresponding auronols (14), Fcr NADH-dependently forms hydroxydihydrochalcones from (2S,3S)-flavanonols. However, the naturally occurring flavanonols exist in their (2R,3R)form, which at least has been demonstrated for taxifolin (23,41,42).…”

“…Besides acting on flavanones, CHI was shown to catalyze the ring contraction of flavanonols (e.g., taxifolin) to isomeric auronols (e.g., alphitonin) ( Fig. 1B) (14).…”

A New Perspective on the Health Benefits of Moderate Beer Consumption: Involvement of the Gut Microbiota

“…This molecule can be metabolized by gut microbiota to render 8-prenylnaringenin [72,73] through an O-demethylation that is carried out by Eubacterium species and particularly by Eubacterium limosum or Eubacterium ramulus that is responsible for this transformation [75]. The genus Eubacterium is one of the biggest butyric acid producers [87] and deserves further investigation. However, little attention has been paid to other hops compounds such as alpha- and beta-acids and their interaction with gut microbiota.…”