Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L
| Autor: | Ulrich Matern, Laura Burga, Simone Witte, Wilfried Schwab, Frank Wellmann, Joachim Schröder, Richard Lukačin |
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| Rok vydání: | 2005 |
| Předmět: |
inorganic chemicals
S-Adenosylmethionine Stereochemistry Cations Divalent Ruta graveolens Molecular Sequence Data Biophysics Phloroglucinol Orcinol Biochemistry Binding Competitive Cofactor Dithiothreitol Substrate Specificity chemistry.chemical_compound Escherichia coli Amino Acid Sequence Cloning Molecular Molecular Biology Cells Cultured chemistry.chemical_classification Ruta biology Chemistry Temperature Methyltransferases Hydrogen-Ion Concentration biology.organism_classification O-methyltransferase Amino acid carbohydrates (lipids) Molecular Weight Kinetics Enzyme Metals biology.protein Thiol |
| Zdroj: | Archives of biochemistry and biophysics. 440(1) |
| ISSN: | 0003-9861 |
| Popis: | A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols. |
| Databáze: | OpenAIRE |
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