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Applied and Environmental Microbiology, September 2003, p. 5089-5095, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5089-5095.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Juan-José R. Coque,^* María Luisa Álvarez-Rodríguez, and Germán Larriba

Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain

Received 21 February 2003/ Accepted 6 June 2003

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-³H]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 µM, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a K_i of 378.9 ± 45.4 µM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu²⁺, Hg²⁺, Zn²⁺, 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.

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* Corresponding author. Mailing address: Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06071 Badajoz, Spain. Phone and fax: 34-924-289424. E-mail: jjrubio{at}unex.es.

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Applied and Environmental Microbiology, September 2003, p. 5089-5095, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5089-5095.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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