Figure 3.

Demethylation of methylcob(III)alamin (MeCbl(III)) catalyzed by purified core-MetE_CBDB from D. mccartyi strain CBDB1 in the presence of D,L-homocysteine. (a) In the presence of 0.1 µM core-MetE_CBDB, 0.5 mM methylcob(III)alamin and 2 mM D,L-homocysteine, continuous changes in the UV/Vis spectrum were observed indicating the consumption of methylcob(III)alamin (524 nm) and the formation of cob(I)alamin (681 nm) and cob(II)alamin (474 nm) as highlighted by arrows. (b) In the absence of core-MetE_CBDB, the UV/Vis spectrum remained unchanged over an incubation time of 30 min. (c) In the absence of D,L-homocysteine, the UV/Vis spectrum remained unchanged. (d) In the presence of 0.5 mg mL⁻¹ E. coli crude extract, instead of core-MetE_CBDB as catalyst, MeCbl(III) was not transformed. (e) Dependence of core-MetE_CBDB methyltransferase activity on methylcob(III)alamin concentration. (f) Dependence of core-MetE_CBDB methyltransferase activity on D,L-homocysteine concentration. The activities in panels (e,f) were determined by following the change of the absorption at 681 nm. V_max and K_M were calculated according to a Hill-Fit plot with R² = 0.998 for panel (e) and R² = 0.999 for panel (f), respectively.