Fig. 4. O-demethylation and methyl transfer conducted by Mto proteins.

Reaction A: MtoD, ATP and titanium (III) citrate are required for activation of MtoC from the Co(II) state (blue) to the active Co(I) state (dark green). Reaction B: MtoB transfers the methyl group of the methoxy compound (Ar-O-CH₃) to Co(I)-MtoC resulting in methylated Co(III)-MtoC (red). The MtoB activities with 2-methoxybenzoate (MB) and TMB are shown in Fig. S2A. With methanol or trimethylamine as substrate no activity could be observed. Conversion of TMB to 3-OH-4,5-dimethoxybenzoate was confirmed by HPLC (Fig. S3). Reaction C: For measuring MtoA activity, the H₄MPT structural analog H₄F was used. We got strong evidence that MtoA transfers the methyl group from methylated Co(III)-MtoC (red) to H₄F thereby producing Co(I)-MtoC (light green). The activity is shown in Fig. S2B. In M. shengliensis H₄MPT and not H₄F is most likely the methyl group acceptor as M. shengliensis does not have the genomic capacity to synthesize H₄F. Also, the methyl-transfer reaction is not occurring if CoM is used instead of H₄F (Fig. S2B). All bottom panels correspond to UV/visible spectra measured after each reaction reflecting the different states of the cobalamin carried by MtoC.