Functional Characterization of Epitheaflagallin 3-O-Gallate Generated in Laccase-Treated Green Tea Extracts in the Presence of Gallic Acid.

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.

Identification of a product specific beta-amyrin synthase from Arabidopsis thaliana.

Triterpene skeletons are produced by oxidosqualene cyclases (OSCs). The genome sequencing of Arabidopsis thaliana revealed the presence of thirteen OSC homologous genes including At1g78950, which has been revised recently as two independent ORFs, namely At1g78950 and At1g78955. The cDNA corresponding to the revised At1g78950 was obtained by RT-PCR, ligated into Saccharomyces cerevisiae expression vector pYES2, and expressed in a lanosterol synthase deficient S. cerevisiae strain. LC-MS and NMR analyses of the accumulated product in the host cells showed that the product of At1g78950 is beta-amyrin, indicating that At1g78950 encodes a beta-amyrin synthase (EC 5.4.99.-).

Origin of structural diversity in natural triterpenes: direct synthesis of seco-triterpene skeletons by oxidosqualene cyclase.

At1g78500, one of the oxidosqualene cyclase (OSC) homologues from Arabidopsis thaliana, was expressed in a lanosterol synthase-deficient yeast strain and the products were analyzed. In addition to the known triterpenes, this OSC was found to produce two new triterpenes, the structures of which were determined by NMR and MS analyses. The new triterpenes are C-ring-seco-beta-amyrin (1) and C-ring-seco-alpha-amyrin (2) and named beta-seco-amyrin and alpha-seco-amyrin, respectively. beta-seco-Amyrin is produced from the oleanyl cation through bond cleavage between C8 and C14, and alpha-seco-amyrin is produced from the ursanyl cation in the same manner. Together with Grob fragmentation catalyzed by another OSC (marneral synthase) from A. thaliana, the formation of seco-amyrins by this OSC revealed that OSCs not only catalyze carbon-carbon bond formations and Wagner-Meerwein rearrangements but also cleave preformed ring systems in cationic intermediates. Based on this information, direct production of other natural seco-triterpenes by OSCs is proposed.

A new triterpene synthase from Arabidopsis thaliana produces a tricyclic triterpene with two hydroxyl groups.

[structure: see text] Thirteen oxidosqualene cyclase homologues exist in the genome of Arabidopsis thaliana. One of these genes, At4g15340, was amplified by PCR and expressed in yeast. The yeast transformant accumulated tricyclic triterpene, (3S,13R)-malabarica-17,21-dien-3,14-diol (arabidiol), whose structure was determined by NMR and MS analyses. Its epoxide analogue, (3S,13R,21S)-malabarica-17-en-20,21-epoxy-3,14-diol (arabidiol 20,21-epoxide), was also isolated from the transformed yeast. This is the first example of a triterpene synthase that yields a tricyclic triterpene with two hydroxyl groups.

Lanosterol synthase in dicotyledonous plants.

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.

Identification of beta-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay.

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.