Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols.

A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes.

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (-)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (-)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC-MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells.

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H(2)O(2)-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3'-O-gallate (TF3'G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3'G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H(2)O(2)-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3'G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p < 0.05), and improved to approximately 100%. Only TF3'G did not significantly increase cell viability. It was suggested that the inhibitory effect on H(2)O(2)-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.

O-methylated theaflavins suppress the intracellular accumulation of triglycerides from terminally differentiated human visceral adipocytes.

A known O-methylated theaflavin, theaflavin 3-O-(3-O-methyl)gallate (3MeTF3G), and the new theaflavin 3-O-(3,5-di-O-methyl)gallate (3,5diMeTF3G) were synthesized via the O-methylation of theaflavin 3-O-gallate (TF3G). Both 3MeTF3G and 3,5diMeTF3G are more stable than TF3G at pH 7.5 in the order 3,5diMeTF3G > 3MeTF3G > TF3G. The inhibitory effects of these compounds on the intracellular accumulation of triglycerides from terminally differentiated human visceral adipocytes were investigated. Compound 3MeTF3G exhibited an inhibitory effect similar to that of TF3G at 3 µM and a slightly lower effect than that of TF3G at 10 µM. The result suggested that the degradants and oxidatively polymerized products of TF3G may also have inhibitory effects. For cells treated with 3,5diMeTF3G at 3 and 10 µM, intracellular triglyceride accumulation was dose dependent and significantly lower compared with that for other compounds. It was suggested that the higher effect of 3,5diMeTF3G was due to its higher stability and likely improved absorption owing to di-O-methylation.

Cloning of a novel O-methyltransferase from Camellia sinensis and synthesis of o-methylated EGCG and evaluation of their bioactivity.

The gene of a novel O-methyltransferase was isolated from tea cultivars (Camellia sinensis L.). Using the recombinant enzyme, O-methylated (-)-epigallocatechin-3-O-gallate (EGCG) in all cases were synthesized. EGCG and the synthesized O-methylated EGCGs including (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3''Me), (-)-epigallocatechin-3-O- (4-O-methyl)-gallate(EGCG4''Me), (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3'',5''diMe), and (-)-3-O-methyl-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3',3'',5''triMe) were assayed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and antibacterial activity. EGCG was the most effective of the O-methylated EGCGs. The antiallergic effects of EGCG and the other O-methylated EGCGs were measured by conducting histamine release assays using bone marrow-derived mouse mast cells, and the order of potency was EGCG3',3'',5''triMe = EGCG3'',5''diMe > EGCG3''Me > EGCG. These results indicated that reducing the number of hydroxyl groups decreases the effectiveness of DPPH radical scavenging and antibacterial activity. In contrast, the inhibition of histamine release was potentiated by an increase in the number of methyl groups in EGCG, especially in the galloyl moiety.