Functional characterization of two p-coumaroyl ester 3′-hydroxylase genes from coffee tree: Evidence of a candidate for chlorogenic acid biosynthesis

Laboratoire de Génomique et Qualité du café, IRD, UMR 1097 DGPC, 911 Avenue Agropolis, BP 64501, 34394, Montpellier cedex 5, France.
Plant Molecular Biology (Impact Factor: 4.26). 06/2007; 64(1-2):145-59. DOI: 10.1007/s11103-007-9141-3
Source: PubMed


Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

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    • "diCQAs and triCQAs also accumulate in tomato fruit (diCQAs, approximately 2 mg 100 g –1 DW; and triCQAs, 1–2 mg 100 g –1 DW; Chanforan et al., 2012). Three pathways (Villegas and Kojima, 1986; Hoffmann et al., 2003; Niggeweg et al., 2004) have been proposed for the synthesis of CGA: (1) the direct pathway involving caffeoyl-CoA transesterification with quinic acid by hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT; Niggeweg et al., 2004; Comino et al., 2009; Menin et al., 2010; Sonnante et al., 2010); (2) the route by which p-coumaroyl-CoA is first transesterified with quinic acid via hydroxycinnamoyl- Coenzyme A transferase (HCT) acyltransferase (Hoffmann et al., 2003; Comino et al., 2007), followed by the hydroxylation of p-coumaroyl quinate to 5-caffeoylquinic acid, catalyzed by C39H (p-coumaroyl-3-hydroxylase; Schoch et al., 2001; Mahesh et al., 2007; Moglia et al., 2009); and (3) the use of caffeoyl-glucoside as the acyldonor (Villegas and Kojima, 1986). In tomato, the synthesis of CGA involves transesterification of caffeoyl-CoA with quinic acid by HQT (Niggeweg et al., 2004). "
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    ABSTRACT: Tomato, like other Solanaceous species, accumulates high levels of anti-oxidant caffeoylquinic acids (CQAs), which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid, CGA) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked anti-oxidative properties, thus they are of therapeutic interest both as 'phytonutrients' in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis, and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by LC-MS as diCQAs. This chlorogenate:chlorogenate transferase (CCT) activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity, and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in E. coli confirmed its ability to synthesize diCQAs in vitro. This second activity (CCT) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localises to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species the enzyme catalyses different reactions in two sub-cellular compartments.
    Plant physiology 10/2014; 166(4). DOI:10.1104/pp.114.251371 · 6.84 Impact Factor
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    • "Z. mays and S. bicolor contain putative orthologs of PvHCT-Like1 (Fig. 1). We also performed a phylogenetic analysis of the encoded protein sequences of PvC3′H1, PvC3′H2, its putative orthologous proteins from S. bicolor and Z. mays, and previously characterized C3′Hs from C. canephora (Mahesh et al. 2007), A. thaliana (Schoch et al. 2001), C. cardunculus (Moglia et al. 2009) and Triticum aestivum (Morant et al. 2007) (Supplemental Fig. S2). The two switchgrass C3′Hs exhibit 78 % similarity. "
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    ABSTRACT: Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.
    Plant Molecular Biology 11/2013; 84(4). DOI:10.1007/s11103-013-0152-y · 4.26 Impact Factor
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    • "The general phenylpropanoid-and monolignol-specific pathways also provide hydroxycinnamic acids, which include p-coumaric, caffeic, ferulic and sinapic acids. Hydroxycinnamic acids can be esterified or amidated by a variety of moieties such as malate, quinate, glucose, sucrose, choline, putrescine, spermidine, hydroxyanthranilate and tyramine; this can differ between plant species and plant tissues (Dimberg et al., 1993; Martin-Tanguy, 1997; Schmidt et al., 1999; Mahesh et al., 2007; Milkowski & Strack, 2010). In Arabidopsis, the largest portion of the ferulic and sinapic acid pool is made from coniferaldehyde and sinapaldehyde via hydroxycinnamaldehyde dehydrogenase (HCALDH) (Nair et al., 2004). "

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