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. 1993 Mar;59(3):706–711. doi: 10.1128/aem.59.3.706-711.1993

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

Catherine Coulter 1, James T Kennedy 2, W Colin McRoberts 2, David B Harper 1,2,*
PMCID: PMC202178  PMID: 16348886

Abstract

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The Km values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.

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Selected References

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  1. AXELROD J., TOMCHICK R. Enzymatic O-methylation of epinephrine and other catechols. J Biol Chem. 1958 Sep;233(3):702–705. [PubMed] [Google Scholar]
  2. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ebel J., Hahlbrock K., Grisebach H. Purification and properties of an o-dihydricphenol meta-O-methyltransferase from cell suspension cultures of parsley and its relation to flavonoid biosynthesis. Biochim Biophys Acta. 1972 May 12;268(2):313–326. doi: 10.1016/0005-2744(72)90326-9. [DOI] [PubMed] [Google Scholar]
  4. Harper D. B., Buswell J. A., Kennedy J. T., Hamilton J. T. Chloromethane, Methyl Donor in Veratryl Alcohol Biosynthesis in Phanerochaete chrysosporium and Other Lignin-Degrading Fungi. Appl Environ Microbiol. 1990 Nov;56(11):3450–3457. doi: 10.1128/aem.56.11.3450-3457.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Harper David B., Hamilton John T. G., Kennedy James T., McNally Kieran J. Chloromethane, a Novel Methyl Donor for Biosynthesis of Esters and Anisoles in Phellinus pomaceus. Appl Environ Microbiol. 1989 Aug;55(8):1981–1989. doi: 10.1128/aem.55.8.1981-1989.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. MANN J. D., FALES H. M., MUDD S. H. ALKALOIDS AND PLANT METABOLISM. VI. O-METHYLATION IN VITRO OF NORBELLADINE, A PRECURSOR OF AMARYLLIDACEAE ALKALOIDS. J Biol Chem. 1963 Nov;238:3820–3823. [PubMed] [Google Scholar]
  8. Poulton J. E., Hahlbrock K., Grisebach H. O-Methylation of flavonoid substrates by a partially purified enzyme from soybean cell suspension cultures. Arch Biochem Biophys. 1977 Apr 30;180(2):543–549. doi: 10.1016/0003-9861(77)90071-6. [DOI] [PubMed] [Google Scholar]
  9. Valli K., Gold M. H. Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J Bacteriol. 1991 Jan;173(1):345–352. doi: 10.1128/jb.173.1.345-352.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Valli K., Wariishi H., Gold M. H. Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium. J Bacteriol. 1992 Apr;174(7):2131–2137. doi: 10.1128/jb.174.7.2131-2137.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Walsh C. Fluorinated substrate analogs: routes of metabolism and selective toxicity. Adv Enzymol Relat Areas Mol Biol. 1983;55:197–289. doi: 10.1002/9780470123010.ch3. [DOI] [PubMed] [Google Scholar]
  12. Wengenmayer H., Ebel J., Grisebach H. Purification and properties of a S-adenosylmethionine: isoflavone 4'-O-methyltransferase from cell suspension cultures of Cicer arietinum L. Eur J Biochem. 1974 Dec 16;50(1):135–143. doi: 10.1111/j.1432-1033.1974.tb03881.x. [DOI] [PubMed] [Google Scholar]

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