Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa

Abstract

Background: Flavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis.

Results: In this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg(2+)-independent O-methyltransferases and showed high amino acid sequence similarity (60-95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-L-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3',5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids.

Conclusions: Five FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3',5,7-tetramethylated quercetin was obtained as the final product.

Keywords: Citrus depressa; O-methyltransferase; S-adenosyl-L-methionine dependent; flavonoid; nobiletin.

Fig. 1

Multiple sequence alignment of CdFOMT sequences with higher plant flavonoid O-methyl transferases (FOMTs): C. depressa (CdFOMT1, DDBJ accession number LC126056; CdFOMT3, LC126057; CdFOMT4, LC126058; CdFOMT5, LC126059; and CdFOMT6, LC126060), Citrus sinensis (CsFOMT, GenBank accession number ABP94018), Arabidopsis thaliana (At3OMT, AED96460), Chrysosplenium americanum (Ca3′OMT1, AAA80579), Mentha × piperita (Mp3′OMT, AAR09601), and Triticum aestivum (Ta3′4′5′OMT, ABB03907). Amino acid sequences were aligned using ClustalW. Black highlighting denotes amino acids that are conserved across many FOMTs

Fig. 2

A phylogenetic tree constructed from plant FOMT amino acid sequences. In addition to sequences of CdFOMTs characterized in this paper, 25 plant class II FOMT sequences were also selected using BLAST search and aligned by ClustalW. The phylogenetic tree was constructed using the NJplot program. Arabidopsis thaliana (At3OMT, GenBank accession number AED96460 and AtOMT1, AAB9679), Catharanthus roseus (CrFOMT, AAM97497 and CrOMT6, AAR02419), Chrysosplenium americanum (Ca3′OMT1, AAA80579), Citrus sinensis (CsFOMT, ABP94018), Glycyrrhiza echinata (Ge4′OMT, AB091684 and Ge7IOMT, AB091685), Hordeum vulgare (HvFI7OMT, CAA54616), Lotus japonicus (Lj4′OMT, AB091686), Medicago sativa (Ms3′OMT, AAB46623 and Ms7IOMT, MSU97125), Mentha × piperita (Mp3′OMT, AAR09601; Mp4′OMT, AAR09602; Mp7OMT1A, AAR09598; Mp7OMT1B, AAR09599; and Mp8OMT, AAR09600), Oryza sativa (Os7OMTlike1, BAD29452; Os7OMTlike2, BAD05699; and Os3OMT, BAF22945), Populus trichocarpa (PtFOMT2, EEE86889; PtOMT5, EPR47487; and PtFOMT10, EPR54183), Triticum aestivum (Ta3′4′5′OMT, ABB03907), and Zea mays (ZmCOMT, ABQ58826)

Fig. 3

a CdFOMT5 expression plasmid and b SDS-PAGE analysis of recombinant CdFOMT5 in E. coli transformant cells. Lane (M) contained the molecular weight markers; lane (C) the cell-free extract; lane (U) non-adsorbed fraction of Ni-Sepharose column chromatography; and lane (P) the purified enzyme

Fig. 4

HPLC analysis of quercetin products methylated by CdFOMT5: S, quercetin; P1, 3-O-methylated quercetin; P2, azaleatin/rhamnetin; P3, dimethylated quercetin; and P4, trimethylated quercetin. The peak labeled with ‘x’ was not identified as corresponding to flavonoid derivatives. (See Additional file 2: Figure S2)

Fig. 5

HPLC chromatograms of monohydroxy flavone products methylated by CdFOMT5: S, substrate control; P, authentic compound of methylated product; and S + E, enzyme reaction product

Fig. 6

Bioproduction of polymethylated quercetin using recombinant E. coli carrying CdFOMT5. a The HPLC analysis of the reaction mixture identified five peaks: (1) 3-O-methylated quercetin; (2) isorhamnetin (3′-O-methylated quercetin); (3) azaleatin/tamarixetin; (4) dimethylated quercetin; (5 and 6) trimethylated quercetin; and (7) 3,5,7,3′-tetramethylated quercetin. Peaks labeled with ‘x’ were not identified as quercetin derivatives. Transformant cells harboring empty vector (pET21b) was used as host cell. b Mass spectrometry analysis of purified peak 7 product. c NMR spectrum of purified product (peak 7). d Predicted structure of 3,3′,5,7-O-methylated quercetin