Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O--D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Enzymatic synthesis of perillyl alcohol derivatives and investigation of their antiproliferative activity

The monoterpene perillyl alcohol (POH), an intermediate in the plant terpenoid biosynthetic pathway, has well-established tumor chemopreventive and chemotherapeutic potential. We have previously shown that the primary hydroxyl group of POH is essential for its antitumor and anti-angiogenic activities. In the current study we present the enzymatic synthesis of two POH derivatives with different polar and hydrophobic characteristics, namely perillyl glucoside and perillyl glucoside fatty ester, through a two-step modification. Initial glucosylation of POH on its active hydroxyl group with D-(+)-glucose and subsequent esterification of the perillyl glucoside product with vinyl laurate were carried out using almond β-glucosidase and lipase B from Candida antarctica, respectively, in a low-water system. Optimization of enzymatic reactions was performed to achieve the highest possible conversion yields. The antitumor cell proliferation activity against mouse Lewis lung carcinoma cells was retained in both derivatives, although the perillyl glucoside ester showed greater inhibition than perillyl glucoside. Our results underline the feasibility of enzymatically producing novel bioactive analogs of phytochemicals displaying useful physicochemical properties.     

Microbial transformation of curcumin by Rhizopus chinensis

Abstract

Curcumin (1) is a potent antioxidant and antitumor natural product. In spite of its efficacy and safety, its clinical use is hindered mainly by poor water solubility and bioavailability. Structural modification to introduce hydrophilic functions is a promising approach to resolve this problem. In the present study we first found that curcumin could be efficiently converted into glucosides by filamentous fungi including Rhizopus chinensis IFFI 03043, Absidia coerulea AS 3.3389 and Cunninghamella elegans AS 3.1207. Curcumin 4′-O-β-d-glucoside (2), together with hexahydrocurcumin (3), was isolated from a preparative-scale biotransformation with R. chinensis IFFI 03043 and characterized fully by NMR and MS. A time-course study revealed that curcumin could be efficiently converted into curcumin 4′-O-β-d-glucoside within 8 h when administered at 0.05 mmol L^?1 and the productivity was 57%. Additionally, the biotransformation products of curcumin by different fungal strains were analyzed by LC/MS. At least 15 metabolites were detected, and the predominant biotransformation reaction was glucosylation. This study provides a simple, efficient and less expensive approach for the preparation of curcumin glucosides. The introduction of the glucosyl function might be able to enhance the bioavailability of curcumin.     

Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.     

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.     

Structural basis for acceptor‐substrate recognition of UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

Biotransformation of hydroquinone to arbutin in plant <Emphasis Type="Italic">in vitro</Emphasis> cultures — preliminary results

Cells from Echinacea purpurea (L.) Moench. (Asteraceae), Exacum affine Balf. f. (Gentianaceae), Melittis melissophyllum L. (Lamiaceae), Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. (Rutaceae) agitating cultures perform a biotransformation reaction on exogenously supplied hydroquinone into its β-D-glucoside — arbutin, product with valuable medicinal and cosmetic properties. The maximum content of arbutin (determined by HPLC) in the biomass from investigated cultures is 4.01; 3.44; 1.79; 2.48 and 5.07 g/100 g d.w., respectively. Nothing but Ammi majus L. (Apiaceae) cultures contain trace amounts of the product. Arbutin is accumulated in cells; it is occasionally found in media only in vestigial amounts. In most of the investigated cultures the efficiency of the biotransformation process is about 60 %.