FAD-dependent C-glycoside–metabolizing enzymes in microorganisms: Screening,characterization, and crystal structure analysis

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme “C-glycoside 3-oxidase” named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

Various low–molecular mass plant-derived compounds, such as flavonoids, are glycosylated. Glycosides can be classified as O-, C-, N-, and S- by the manner of linkage between a sugar and aglycone, which is the nonsugar moiety of glycosides (Fig. 1A). Hundreds of C-glycosides have been isolated from various living organisms and show various bioactivities (1–4).Open in a separate windowFig. 1.Schematic representation of O-glycosides and C-glycosides, the growth of Microbacterium sp. 5-2b, and HPLC analysis of reaction mixtures. (A) Structures of O-glycosides (Left) and C-glycoside (Right). The cleavage sites for deglycosylation are indicated by the dotted lines. (B, Left) Colonies of strain 5-2b on a culture plate containing carminic acid, the color of which is red. Strain 5-2b was cultured on this plate for 7 d. (B, Right) Corresponding to B, Left, the colonies of strain 5-2b are colored yellow, and the clear zone is indicated by a white dotted line so that it is easier to discern. (C) HPLC analysis of reaction mixtures; cell-free extracts of strain 5-2b were incubated with carminic acid for 0 min, 10 min, and 2 h. The numbers in this figure indicate carminic acid (1), compounds X₁ and X₂ (2, 3), and compound Y (kermesic acid, 4).Humans ingest these glycosides in botanical foods and metabolize them in the intestine. In the small intestine, lactase-phlorizin hydrolase, which is present on the luminal side of the brush border, hydrolyzes glycosides (5) and, in the large intestine, glycosides are metabolized by intestinal microorganisms (6). The resulting aglycones are taken up from the intestine and show many beneficial bioactivities, such as antimicrobial, antiviral, and antioxidative ones (7). The metabolism of glycosides is closely related to the expression of their biological activities.More than 100 glycoside hydrolase families in the CAZy database which hydrolyze glycosides to yield a sugar moiety and the corresponding aglycone have been identified from bacteria to mammals (8–11). However, C-glycosides are not deglycosylated by glycoside hydrolases, because the sugar moiety and the aglycone are linked by a carbon–carbon bond. In intestinal microorganisms, C-glycosides are deglycosylated through a two-step deglycosylation reaction consisting of oxidation of the sugar moiety and C–C bond cleavage (12–15). Although the enzymes catalyzing the two-step reaction have been identified (15), detailed biochemical characterization and crystal structure analysis have never been reported.To clarify the metabolism of C-glycosides in nature, we started our study by screening carminic acid–catabolizing microorganisms from soil. Carminic acid, which is extracted from cochineal insects, is a C-glycoside of an anthraquinone derivative and is very important in the food, cosmetic, and pharmaceutical industries as a natural “red dye” all over the world (16).In this study, we discovered a carminic acid–catabolizing microorganism and identified a C-glycoside–metabolizing enzyme, C-glycoside 3-oxidase (CarA), that catalyzes the first step of C-glycoside metabolism by oxidizing the C3 position of the sugar moiety. We also report the enzyme’s biochemical properties, crystal structure, and possible reaction mechanism.