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 degrees 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 micro M, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a K(i) of 378.9 +/- 45.4 micro 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.

FIG. 1.

Time course of induction of methyltransferase activity by 2,4,6-TCP (○) in a resting-cell system and activity in noninduced mycelia (▵). The values are estimates resulting from duplicate determinations in three independent experiments. The error bars indicate standard deviations of the means.

FIG. 2.

Induction of CPOMT activity by several CPs, including 2,4-DCP (bar A), 2,6-DCP (bar B), 2,3-DCP (bar C), 3,4-DCP (bar D), 2,4,6-TCP (bar E), 2,4,5-TCP (bar F), 2,3,4,6-TeCP (bar G), and PCP (bar H), and activity of noninduced mycelium (bar I). A value of 100 was arbitrarily assigned to the level of induction obtained with 2,4,6-TCP (bar E). The error bars indicate standard deviations of the means.

FIG. 3.

Electrophoretic analysis of CPOMT. (A) SDS-PAGE of purified CPOMT: Coomassie blue staining of molecular weight standards (lane 1) and the purified enzyme (lane 2). (B) Detection of CPOMT by photolabeling with [³H]SAM. Lanes 1 and 2, Coomassie blue staining of broad-range SDS-PAGE molecular weight standards (lane 1) and the enzyme preparation after step 4 of the purification process (lane 2); lanes 3 and 4, fluorography of the photolabeling reaction mixture with the protein preparation shown in lane 2 in the absence (lane 3) and in the presence (lane 4) of 1 mM SAHC. The position of CPOMT is indicated by an arrow.