Molecular cloning and yeast expression of cinnamate 4-hydroxylase from Ornithogalum saundersiae baker.
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.
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. jianqiangk@imm.ac.cn.
Jian-Qiang
JQ
Kong
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Di
D
Lu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Zhi-Biao
ZB
Wang
D002783:Cholestenones / Q000737:chemistry / Q000302:isolation & purification; D002934:Cinnamates / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D006900:Hydroxylation; D031442:Ornithogalum / Q000201:enzymology*; D018517:Plant Roots / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012503:Saponins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D050564:Trans-Cinnamate 4-Monooxygenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D002783:Cholestenones; D002934:Cinnamates; D018076:DNA, Complementary; D012503:Saponins; C029010:cinnamic acid; C106408:OSW 1; D050564:Trans-Cinnamate 4-Monooxygenase