Biotransformation of Ferulic Acid into Two New Dihydrotrimers by Momordica charantia Peroxidase

Abstract

Previously, we reported the physical and chemical characterization of Momordica charantia peroxidase (MCP), a novel plant peroxidase with high acidic amino acid purified from the fruits of Momordica charantia and applied the purified MCP to transform ferulic acid (FA) into FA-2, a FA dehydrodimer, which had more powerful anti-inflammation than FA. 1) We found that although MCP shared spectral and kinetic features with other peroxidases, the enzyme had several unique characteristics, including enzyme pH stability (pH 3.8-8.0) and thermostability (20-45°C) wider than those of other peroxidases such as horseradish peroxidase. 2)So MCP can be expected to oxidize a wider range of substrates, especially cinnamic acid derivatives, when considering the potential applications of MCP for useful biotransformation.FA, which is an extremely abundant and widespread cinnamic acid derivative, was a good substrate for most plant peroxidases such as lignin peroxidase, wheat germ peroxidase and horseradish peroxidase.3,4) FA dehydrodimers produced both in vitro and in vivo by plant peroxidases have been extensively characterizied.5-11) They result from oxidative coupling of ferulate esters and represent mainly products of 8-5, 8-O-4, 4-O-5 and 5-5 radical couplings. Recently, it was reported that in incubation with lignin peroxidase and ectomycorrhizal fungi, dihydrotrimers of FA were formed.12,13) However, only two structures of FA dehydrotrimers were elucidated by NMR spectrum, although higher oligomers of FA on a template of a tyrosine-containing tripeptide have recently been yielded enzymically in vitro. 14) In order to further study the biotransformation of FA by purified MCP and analyze FA dehydrotrimers, we have investigated the oxidative coupling of FA with H 2 O 2 /MCP at pH 5.0 in the presence of acetone. Two new FA dehydrotrimers, triFA1 (1) and triFA2 (2), have been identified. TriFA1 (1) is a unique FA dehydrotrimer possessing a 3a,9b-dihydro-1H-furo [3,4-c][1]benzopyran-3(4H)-one ring system.In the present paper, we report on the biotransformation of FA by MCP and the isolation and the structural characterization of the two new FA dehydrotrimers, as well as the possible formation mechanism for triFA1 (1). Results and DiscussionTriFA1 (1) As a result of a continuing investigation of the biotransformation of ferulic acid with H 2 O 2 /Momordica charantia peroxidase at pH 5.0 in the presence of acetone, triFA1 (1) and triFA2 (2), two new FA dehydrotrimers, have been isolated. The structures were elucidated from the spectroscopic data including 2D-NMR such as HSQC, HMBC and NOESY data. TriFA1 (1) is a unique FA dehydrotrimer possessing a 3a,9b-dihydro-1H-furo[3,4-c][1]benzopyran-3(4H)-one ring system. The possible mechanism for the formation of triFA1 was also discussed.

Chemistry and occurrence of hydroxycinnamate oligomers

“…The coupling products of the free acids significantly differ from the products obtained from using esters as starting materials (Ward et al 2001). As hydroxycinnamic acids tend to decarboxylate under these conditions (Ward et al 2001;Liu et al 2005Liu et al , 2007a) the coupling products do not represent possible products in the plant cell wall where ferulic acid is ester-linked to cell wall polysaccharides. Esterification, however, protects ferulic acid from being decarboxylated during oxidative coupling.…”

“…As already described for ferulate dehydrodimers, model reactions using the free acids as starting materials are only of limited value to describe processes in the plant cell wall, but might be of interest for other purposes such as demonstrating the detoxification of ferulic acid by fungi. In model reactions using the free acids, ferulic acid (1, R = H), p-coumaric acid (2, R = H), sinapic acid (3, R = H) and caffeic acid 15 were coupled by using different peroxidases and coupling conditions (Ward et al 2001;Liu et al 2005Liu et al , 2007a; Monien et al 2006). Kong's group used peroxidase from Momordica charantia and H 2 O 2 in a buffer (pH 5.0)/acetone mixture to oligomerize p-coumaric, caffeic, ferulic, and sinapic acid and described this in four publications (Liu et al 2005(Liu et al , 2007aWan et al 2008 (Ward et al 2001;Liu et al 2005).…”

“…Compounds 21 and 22 are similar, but not identical. Compound 22 is additionally acetylated, a fact which is not further discussed in the publication (Liu et al 2005).…”

“…Decarboxylated dimers are involved in the formation of these trimeric structures(Ward et al 2001, Liu et al 2005. b Dehydrotri (TriFA) and tetraferulates (TetraFA) as their ethyl esters isolated from a model system using ethyl ferulate as starting material and peroxidase/H 2 O 2 as the oxidizing system.…”

Biotransformation of p‐Coumaric Acid (=(2E)‐3‐(4‐Hydroxyphenyl)prop‐2‐enoic Acid) by Momordica charantia Peroxidase

“…-Previously, we reported the physical and chemical characterization of Momordica charantia peroxidase (MCP), a novel plant peroxidase, rich in acidic amino acid, isolated from the fruits of Momordica charantia [1]. We found that although MCP shared spectral and kinetic features with other peroxidases, the enzyme had several unique characteristics, including enzyme pH stability (pH 3.8 -8.0) and a wider thermostability (20 -458) than that of other peroxidases such as horseradish peroxidase [2]. So, MCP can be expected to oxidize a broader range of substrates, especially cinnamic acid derivatives, when considering the potential applications of MCP for useful biotransformations.…”