Fig 1.
Methyltransfer catalyzed by the C-methyltransferases CouO and NovO.
The alkylation of aromatic substrates with coumarin and naphthalene scaffold is shown. The enzymes also accept SAM-analogues to yield allyl-, propargyl- and benzyl-arenes [3].
Table 1.
CouO structure refinement and validation statistics.
Fig 2.
Overall structures of CouO and similar methyltransferases.
Monomers of A) CouO, B) Coq5 (PDB: 4obw), C) mmp1179 (PDB: 3dlc), D) YexO (PDB: 1im8) and CmoA (PDB: 4gek) are shown in ribbon representations. The cofactors (SAH in CouO, SAM in Coq5 and mmp1179, Cx-SAM in CmoA and SAI in YecO) are shown in sticks representations and the active site cavities are shown as surfaces. The cavity surfaces are colored according to their hydrophobicity (red-hydrophobic to blue-hydrophilic). The main differences are observed in the conformation of the cap-domain (violet) and in the position of the N-terminal part consisting of a loop and the first α-helix (salmon).
Fig 3.
A) Amino acid residues and SAH cofactor in the active site of CouO. B) The lowest energy docking mode (as calculated using the program YASARA) places the coumarin moiety (in cyan) in the vicinity of the SAM cofactor. Amino acid residues His120 and Arg121 are situated about 3 Å from the hydroxyl-oxygen at C-7 of the substrate. These interactions are indicated as yellow dashed lines.
Fig 4.
π-stacking interactions between side chains in the active site.
The interaction between His120 and Phe147 very likely ensures the optimal orientation of the imidazole group of the histidine for the deprotonation of the substrate hydroxyl group. The coumarin moiety (placed by molecular docking calculations) is show in cyan. Hydrogen bonding interactions are indicated by dashed yellow lines.
Fig 5.
Sequence comparison of NovO and CouO.
Identical residues are shown in red boxes. Secondary structure elements of CouO are shown in blue above the sequence alignment. Important amino acid residues in the active site are marked with an asterisk and shown in cyan (identical) and green (similar) boxes. This figure was prepared using ESPript 3.0 [20].
Fig 6.
Reaction mechanism for the Friedel–Crafts alkylation catalyzed by SAM-dependent C-methyltransferases, CouO from Streptomyces rishiriensis and NovO from Streptomyces spheroides.
Fig 7.
Relative activities of the CouO variants.
The individual activities of the variants in the methylation of the coumarin compound N-(4,7-dihydroxy-2-oxo-2H-chromen-3-yl)benzamide (educt) are expressed as % product formation relative to the wild-type enzyme. The reported values are mean values from triplicate measurement and the bars indicate the minimum and maximum values obtained.