Figure 4.

Core-MetE_CBDB from D. mccartyi strain CBDB1 does not catalyze the formation of L-methionine with 5-methyl-THF-Glu₃ as the methyl donor. (a) Reaction described for tr-MetE from E. coli (tr-MetE_Eco) catalyzing the methylation of L-homocysteine with 5-methyl-THF-Glu₃ to form L-methionine and THF-Glu₃⁸⁰. (b) Representative HPLC chromatograms of a chemical standard of 5-methyl-THF-Glu₃ (blue), of reaction products after an enzyme activity assay containing 5-methyl-THF-Glu₃, D,L-homocysteine and either core-MetE_CBDB (red) or tr-MetE_Eco (green) or no enzyme (black). The peak at RT = 21 min represents dithiothreitol added to all reactions. In the presence of tr-MetE_Eco, 5-methyl-THF-Glu₃ (RT = 18 min) reacts to form THF-Glu₃ (RT = 17.4 min). Slow demethylation of 5-methyl-THF-Glu₃ to THF-Glu₃ did occur in the negative control and also in the presence of core-MetE_CBDB. To evaluate if this demethylation was linked to L-methionine formation, the products of the activity assays were analyzed by mass spectrometry. I) No L-methionine was formed in the presence of core-MetE_CBDB; II) The product of tr-MetE_Eco was identified as L-methionine ([M + H]+  = 150.0583 m/z).