FIG 6.

In vitro estrone methylation using l-methionine, methyl-¹³C-l-methionine, or l-serine as methyl donors for SAM regeneration. (A) UPLC analysis of the conversion of estrone (0.1 mM) to ADD by cell extracts of D. oestradiolicum in the presence of 0.5 mM SAM (black line), 0.5 mM l-methionine and 1 mM ATP (red line), or 0.5 mM l-serine, 1 mM ATP, 0.5 mM THF, and 0.5 mM l-homocysteine (green line). (B) ESI-Q-TOF analysis of the ADD product formed in the in vitro assays shown in (A) and in the presence of SAM, methionine (Met), methyl-¹³C-l-methionine (¹³C-Met), and serine. (C) ESI-Q-TOF analysis of the methylcob(III)alamin intermediate in the presence of Met and ¹³C-Met. Due to the doubly charged ions, an m/z shift of 0.5 was detected in in the assay with ¹³C-Met; the m/z = 664.79 peak is assigned to demethylated methylcob(III)alamin.