Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

ABSTRACT The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolinto the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacteriumramulus was purified and characterized. It stereospecificallycatalyzed the isomerization of (+)- taxifolinbut not that of (−)- taxifolin. The K m for (+)- taxifolinwas 6.4 ± 0.8 μM, and the V max 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 flavanonolsand their stereospecificconversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonolisomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecificabstraction of a proton from the hydroxymethylene carbon of the flavanonolC-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 isomerasewas known to be involved in flavone/ flavanoneconversion 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 isomeraseplays 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 isomerasein flavonoid-degrading bacteria.