Characterization of an Inducible Chlorophenol O -Methyltransferase from Trichoderma longibrachiatum Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines

Abstract

A novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3 H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the M r was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The K m values for 2,4,6-trichlorophenol and SAM were 135.9 ؎ 12.8 and 284.1 ؎ 35.1 M, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a K i of 378.9 ؎ 45.4 M. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu 2؉ , Hg 2؉ , Zn 2؉ , and Ag ؉ , and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.

Molecular Fingerprinting by PCR-Denaturing Gradient Gel Electrophoresis Reveals Differences in the Levels of Microbial Diversity for Musty-Earthy Tainted Corks

“…Methylating reactions have been confirmed with a partially purified chlorophenol O-methyltransferase from Trichoderma longibrachiatum isolated from cork (3,13). In addition, the participation of bacteria, mainly Streptomyces spp.…”

Biodegradation kinetics and mechanism of 2,4,6-tribromophenol by Bacillus sp. GZT: A phenomenon of xenobiotic methylation during debromination

“…In addition, most research works have preferred S-adenosyl-L-methionine (SAM), which was produced from methionine (Met), to be the intermediary to induce the reaction of xenobiotic methylation through the Met cycle (Finkelstein, 1990;Reed et al, 2004). Recent studies showed that some halogenated phenols also could induce the SAM-dependent methyltransferase to catalyze o-methylation (Neilson et al, 1988;AlvarezRodriguez et al, 2002;Coque et al, 2003). Accordingly, in this work, it can be proposed that during the TBP methylation, the methyl originating from the formic acid was transferred to the xenobiotic substrate TBP via the Met cycle, and the reactions including the o-methylation and the debromination co-occurred during the xenobiotic metabolism (Neilson et al, 1988;Harper et al, 1989;AlvarezRodriguez et al, 2002;Coque et al, 2003).…”

Regiospecific O-Methylation of Naphthoic Acids Catalyzed by NcsB1, an O-Methyltransferase Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

“…Methyltransferases requiring divalent cations for optimal activity are known (33)(34)(35)(36)(37)(38), and we next evaluated the activity of NcsB1 in the presence of different metal ions. Notably, the presence of EDTA resulted in a 2-fold higher activity; inclusion of Mn 2ϩ and Ca 2ϩ in the reactions resulted in a slightly higher activity, whereas inclusion of Mg 2ϩ afforded a slight decrease in enzyme activity.…”