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Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook. and Arn.

ABSTRACT: Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-?-glucoside]-5-O-[6-O-(malonyl)-?-glucoside], apigenin-7-O-?-glucoside-4'-O-(6-O-malonyl)-?-glucoside, and Mg2+ and Fe3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded 61,491 contigs. These were searched using BLAST against petunia or torenia flavonoid biosynthetic proteins, which identified 11 putative full-length protein sequences belonging to the flavonoid biosynthetic pathway. RT-PCR using primers designed on the basis of these sequences yielded 14 sequences. Spatio-temporal transcriptome analysis indicated that genes involved in the early part of the pathway are strongly expressed during early-petal development and that those in the late part at late-flower opening stages, but they are rarely expressed in leaves. Flavanone 3-hydroxylase and flavonoid 3',5'-hydroxylase cDNAs were successfully expressed in yeast to confirm their activities. Recombinant anthocyanin O-methyltransferase cDNA (NmAMT6) produced using Escherichia coli was subjected to biochemical characterization. Km of NmAMT6 toward delphinidin 3-O-glucoside was 22?µM, which is comparable with Km values of anthocyanin O-methyltransferases from other plants. With delphinidin 3-O-glucoside as substrate, NmAMT6 almost exclusively yielded petunidin 3-O-glucoside rather than malvidin 3-O-glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.

SUBMITTER: Okitsu N

PROVIDER: S-EPMC6543731 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook. and Arn.

Okitsu Naoko N Mizuno Takayuki T Matsui Keisuke K Choi Sun Hee SH Tanaka Yoshikazu Y

Plant biotechnology (Tokyo, Japan) 20180328 1

Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside], apigenin-7-O-β-glucoside-4'-O-(6-O-malonyl)-β-glucoside, and Mg2+ and Fe3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded ...[more]

PMID: 31275032

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Project description:BackgroundFlavonoids 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.ResultsIn 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.ConclusionsFive 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.

Dataset Information

Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook. and Arn.

Publications

Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin <i>O</i>-methyltransferase of <i>Nemophila menziesii</i> Hook. and Arn.

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