Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L.

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 K-m 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 K-i of 52.0 mu M. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols. (c) 2005 Elsevier Inc. All rights reserved.