FIGURE 3.

Characterization of NcsB1 as an O-methyltransferase in vitro. A, NcsB1-catalyzed regiospecific methylation of 2,7-dihydroxy-5-methyl-1-naphthoic acid (8) to afford 2-hydroxy-7-methoxy-5-methyl-1-naphthoic acid (9); B, HPLC analyses of (I) the incubation of 8 with AdoMet alone or 8 and AdoMet with boiled NcsB1 at 25°C for 1 h; (II) incubation of 8 with NcsB1 and AdoMet at 25 °C for 1 h; (III) authentic standard of product 9; and (IV) incubation of 8 and NcsB1 without AdoMet at 25 °C for 2 h ((♦) 8 and (•) 9); C, kinetic characterization of NcsB1 by plotting the initial velocities of 9 formation as a function of 8 (I) at a saturating concentration of AdoMet or AdoMet (II) at a saturating concentration of 8.