Plant methionine synthase: new insights into properties and expression.
We investigated the enzyme methionine synthase (MSY) in Catharanthus roseus. The properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in Escherichia coli. The protein was a monomer and accepted both the triglutamate (CH3-H4PteGlu3, apparent Km = 80 microM) and the monoglutamate (CH3-H4PteGlu1, apparent Km = 350 microM) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90:1 in favor of the triglutamate. Both activities required inorganic phosphate, but with different kinetics, and both were dependent on reducing agents. The activity required zinc, as shown by depletion and reconstitution experiments. Mg2+ had no effect on the activity. Two MSY isoforms purified from parsley cell cultures revealed the same properties as the C. roseus enzyme, however, the parsley proteins had no detectable activity with the monoglutamate substrate. The second part of the work compared the expression of the three enzymes of the methyl cycle (MSY, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase). In cell cultures, all three enzymes were present under all conditions investigated, with small changes at the protein level and more pronounced changes at the RNA level. Studies with seedlings revealed a low expression of all three enzymes in cotyledons, when compared to hypocotyls and radiculas. Immunohistochemical experiments indicated that MSY expression in cotyledons is cell-type specific, with the strongest signals detected in the upper epidermis.
Eckermann
C
C
Institut für Biologie II, Universität Freiburg, Germany.
Eichel
J
J
Schröder
J
J
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D043383:Adenosylhomocysteinase; D002413:Cations, Divalent / Q000494:pharmacology; D018929:Cell Culture Techniques; D018548:Cotyledon / Q000737:chemistry; D006867:Hydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000378:metabolism; D008745:Methylation; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014018:Tissue Distribution; D014280:Triglycerides / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D014280:Triglycerides; D012313:RNA; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D008716:Methionine Adenosyltransferase; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D015032:Zinc