Abstract
OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5′ non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-MS and nuclear magnetic resonance (NMR) analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.
Highlights
OSW-1 (3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-{O-[2-O-(4-methoxybenzoyl)-β-D-xylopyranosyl]-(1→3)-2-O-acetyl-α-L-arabinopyranoside}, Figure 1), was isolated from the bulbs of Ornithogalum saundersiae Baker (O.saundersiae) a perennial plant of the lily family cultivated in Southern Africa [1,2]
OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-MS
Biogenetic analysis showed there were at least five kinds of enzymes responsible for OSW-1 biosynthesis, that is steroid pathway enzymes resulting in OSW-1 aglycone formation, P450 hydroxylase able to add hydroxyl groups to the positions 3, 16 and 17 of the OSW-1 aglycone, a glycosyltransferase involved in disaccharide moiety attachment to the 16-OH of the OSW-1 aglycone, acyltransferases catalyzing introduction of the acetyl and the 4-methoxybenzoyl groups on the disaccharide moiety, and phenylpropanoid biosynthetic pathway enzymes converting aromatic amino acids to a 4-methoxybenzoyl group (Figure 1)
Summary
OSW-1 (3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-{O-[2-O-(4-methoxybenzoyl)-β-D-xylopyranosyl]-(1→3)-2-O-acetyl-α-L-arabinopyranoside}, Figure 1), was isolated from the bulbs of Ornithogalum saundersiae Baker (O.saundersiae) a perennial plant of the lily family cultivated in Southern Africa [1,2]. OSW-1 is about 10-100 times more cytotoxic than clinically applied anticancer agents such as mitomycin C, adriamycin, cisplatin, camptothecin, and paclitaxel, but has a low toxicity towards normal cells [1]. 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by the phenylpropanoid biosynthetic pathway (Figure 1), but molecular biological characterization of related genes has not been fully achieved. Molecular isolation and functional characterization of C4H genes, is an important step in the characterization of the biosynthetic pathway of OSW-1 [11,12,13,14]. A comprehensive and precise functional analysis was carried out This is the first report of gene isolation and functional characterization of OsaC4H from O. saundersiae
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