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 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.
Burga
Laura
L
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
Wellmann
Frank
F
Lukacin
Richard
R
Witte
Simone
S
Schwab
Wilfried
W
Schröder
Joachim
J
Matern
Ulrich
U
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002413:Cations, Divalent; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010696:Phloroglucinol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D032105:Ruta / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D008670:Metals; C015155:phloroglucinol dimethyl ether; D012436:S-Adenosylmethionine; D010696:Phloroglucinol; C505010:3,5-dimethoxyphenol O-methyltransferase, Ruta graveolens; D008780:Methyltransferases