Georg G. Gross

Biosynthesis of gallotannins: formation of polygalloylglucoses by enzymatic acylation of 1,2,3,4,6-penta-O-galloylglucose.

Enzyme preparations from leaves of Rhus typhina L. (sumach) catalyzed the galloylation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose in the presence of the acyl donor beta-glucogallin (1-O-galloyl-beta-D-glucopyranose), yielding a variety of oligomeric gallotannins (hexa- to nonagalloylglucoses) as reaction products.

Biosynthesis of gallotannins : Enzymatic 'disproportionation' of 1,6-digalloylglucose to 1,2,6-trigalloylglucose and 6-galloylglucose by an acyltransferase from leaves of Rhus typhina L.

Cell-free extracts from leaves of Rhus typhina L. (sumach) were found to transfer the 1-O-galloyl moiety of l,6-di-O-galloyl-β-d-glucose to the 2-position of the same compound, yielding 1,2,6-tri-O-galloyl-β-d-glucose and leaving 6-O-galloylglucose as the deacylated by-product. The enzyme catalyzing this ‘disproportionation’ was purified almost 1700-fold. It had a molecular weight of approx. 56 000, a Km value of 11.5 mM, was stable between pH 4.5 and 6.5, and most active at pH 5.9 and 40° C. The systematic name “1,6-di-O-galloyl-glucose: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1.) was proposed for this new enzyme whose detection provided evidence that, in addition to β-glucogallin (1-O-galloyl-β-d-glucose), higher substituted glucose esters also have the potential to serve as acyl donors in the biosynthesis of gallotannins.

Enzymatic synthesis of 1-O-phenylcarboxyl-β-d-glucose esters

Abstract A procedure for the facile preparation of various 1- O -phenylcarboxyl-β- d -glucose esters has been developed. Although such compounds are widely distributed in higher plants, only fragmentary knowledge exists about their synthesis and properties. The method reported here utilizes a UDP-glucose-specific glucosyltransferase from oak leaves. Under the catalysis of this enzyme, several differently ring-substituted 1- O -benzoylglucoses were synthesized. The reaction products were purified by reversed-phase HPLC and characterized by uv, ir, and 1 H NMR spectroscopy. The described procedure appears most suitable for small-scale preparations sufficient for many biochemical investigations, and particularly for the synthesis of radioactively labeled esters.

Biosynthesis of gallotannins. Enzymatic conversion of 1,6-digalloylglucose to 1,2,6-trigalloylglucose

Cell-free extracts from Rhus typhina L. (staghorn sumach) leaves were found to catalyze the transfer of the galloyl moiety of β-glucogallin (1-O-galloyl-β-D-glucose) to 1,6-di-O-galloyl-β-D-glucose, resulting in the specific formation of 1,2,6-tri-O-galloyl-β-D-glucose, an intermediate of gallotannin biosynthesis. The reaction product was unequivocally identified by co-chromatography with authentic references using reversed-phase high-performance liquid chromatography and by 1H-nuclear-magnetic-resonance spectroscopy.

Enzymatic Synthesis of 1,6-Digalloylglucose from β-Glucogallin by β-Glucogallin: β-Glucogallin 6-O-Galloyltransferase from Oak Leaves

Abstract Cell-free extracts from oak (Quercus robur) leaves catalyze the transfer of the galloyl-moiety of β-glucogallin (1-O-galloyl-β-D-glucopyranose) specifically to the 6-position of the same compound, yielding 1,6-di-O-galloyl-β-D-glucopyranose, an intermediate of gallotannin biosynthesis, β- Glucogallin thus functions as both donor and acceptor molecule in this reaction. The partial purification and some general properties of this new acyltransferase are reported.

Immunocytochemical studies on the origin and deposition sites of hydrolyzable tannins.

Two specific antibodies, directed toward hydrolyzable tannins, i.e. polygalloylated or oxidized derivatives of 1,2,3,4,6-penta-O-galloylglucose, and toward an acyltransferase from oak leaves that catalyzes the biosynthesis of this principal precursor, were used in immunocytochemical studies to determine the intracellular sites of origin and deposition of these polyphenolic plant constituents. Immunostaining of semi-thin sections from leaves and roots of young pedunculate oak (Quercus robur, syn. Quercus pedunculata) plants with marker enzymes and immunogold labeling of ultra-thin sections revealed immunoreactive sites for both enzyme and hydrolyzable tannins in chloroplasts, cell walls and intercellular spaces. The latter non-cytoplasmic (apoplast) compartments displayed characteristic aggregations of these two epitopes, thus indicating an intimate association of the biocatalyst and its products. Identical distribution patterns for hydrolyzable tannins were observed in leaves of Rhus typhina (sumac) and Tellima grandiflora (fringe cups) which, however, displayed no affinity toward the galloyltransferase antibody that had been raised against enzyme from oak. Controls with spinach leaves, known to be devoid of tannins, were inactive in all cases. The conclusion that, besides chloroplasts, cell walls and intercellular space serve as sites for the biosynthesis and deposition of hydrolyzable tannins was confirmed by analyzing extracts from these non-cytoplasmic compartments.

β-Glucogallin-Dependent Acyltransferase from Oak Leaves. II. Application for the Synthesis of 1-O-Phenylcarboxyl-β-D-[14C]glucose Esters*

Summary An acyltransferase from young oak ( Quercus robur ) leaves catalyzes a rapid exchange reaction between various l- O -phenylcarboxyl-β-D-glucose esters and free D-[U- 14 C]glucose. As shown for β-glucogallin, this procedure can be used for the convenient preparation of such esters with high specific radioactivity of the labelled carbohydrate moiety. The described method provides an attractive and comparatively economic alternative to conventional chemical procedures.

Biosynthesis Of Gallotannins. ß-Glucogallin-Dependent Galloylation Of 1,6-Digailoylglucose To 1,2,6-Trigalloylglucose

An enzyme from leaves of sumach (Rhus typhina) was partially purified that catalyzes the β-glucogallin (l-O-galloylglucose)-dependent galloylation of 1,6-digalloylglucose, thus forming 1,2,6-trigalloylglucose and free glucose. This acyltransferase had a molecular weight of ca. 750,000 and a pH optimum at 5.0-5 .5. Besides β-glucogallin (Km = 3.9 mM ) , also related 1-O-phenylcarboxylglucoses acted as acyl donors. On the other hand, the acceptor substrate, 1,6-digalloylglucose (Km = 0.9 mM ) , could only be replaced by 1,6-diprotocatechuoylglucose (relative activity 46%); however, also tri-, tetra-, and pentagalloylglucoses were galloylated. A pronounced stimulation of the enzymatic reaction was observed upon addition of penta- or hexagalloylglucose into the assay mixtures. The systematic name “ β-glucogallin: 1,6-di-O-galloylglucose 2-O-galloyltransferase” (EC 2.3.1. - ) is proposed for the enzyme

Synthesis of Galloyl-Coenzyme A Thioester

Galloyl-CoA, a potential intermediate in the biosynthesis of gallotannins, has been prepared via N-succinimidyl 4-O-β-ᴅ-glucosidogallate and 4-O-β-ᴅ-glucosidogalloyl-CoA. Besides a major absorption band at 261 nm, the UV-spectra of the purified thioester and its corresponding 4-O-glucoside contain a longer wavelenght absorption band due to the thioester linkage at 305 nm (galloyl-CoA) or at 290 nm (shoulder, glucosidogalloyl-CoA). The molar extinction coefficients ε of the two thioesters were determined via the iron-complex of 4-O-β-ᴅ-glucosidogalloyl hydroxamic acid; ε261-values of 19.5 × 106 [cm2 mol-1] and 21.5 × 106 [cm2 mol-1] were calculated for galloyl-CoA and its glucoside, respectively. Difference spectra, i.e. absorbance before esterolysis and after, revealed maximal absorption of the thioester bond at 310 nm (⊿ε = 7.4 × 106 [cm2 mol-1]) for galloyl-CoA and at 282 nm (⊿ε = 7.2 × 106 [cm2 mol-6]) for glu- cosidogalloyl-CoA. The two thioesters were further characterized by determining their half-lifes during hydroxylaminolysis and alkaline hydrolysis.

Synthesis of Piperoyl Coenzyme A Thioester

Piperoyl-CoA, a potential intermediate in the biosynthesis of piperine. the pungent principle of pepper, was synthesized via N-succinimidyl piperate and subsequent transesterification with CoA- SH. UV-spectrophotometry of the HPLC-purified compound revealed, besides a maximum at 260 nm, a pronounced second absorption maximum at 368 nm due to the thioester linkage for which, as determined via the Fe3+-complex of piperoyl hydroxamate and with [14C]piperoyl-CoA, a molar extinction coefficient of 30.8 × 106 [cn2mol-1] was calculated.