Extended Data Fig. 3: EPR analysis and activity of Mmp10 and mutants.

a, LC-MS analysis of the reaction catalyzed by Mmp10. SAM [M]⁺= 399.14, 5′-dA [M+H]⁺= 252.11 and SAH [M+H]⁺= 385.13 are indicated. The peptide substrate [M+H]⁺= 1496.77 is converted into a methylated species [M+H] ⁺= 1510.79 (see panel c for full assignment). b, Time course analysis of the reaction catalyzed by Mmp10. c, LC-MS/MS analysis of the 13-mer peptide before (upper panel) and after (lower panel) incubation with Mmp10. See Extended Data Table 2 for full assignment. d, Schematic representation of the A3-mutant, biochemical characterization and EPR analysis. Right upper panel: LC-MS analysis of the reaction before (blue trace) and after (red trace) 1 hr incubation. Right lower panel: Time course analysis of the reaction. Left lower panel: CW EPR analysis. The weak signal measured at g ~ 2.0 represents adventitious FeS cluster. e, Schematic representation, EPR analysis and activity assay of the A4-mutant lacking C35, C38, C45 and C48 involved in the Fe-loop coordination. Upper right panel: CW EPR analysis. Lower panel: LC-MS analysis of the reaction catalyzed by the A4-mutant before (blue trace) and after (red trace) 1 hr incubation. SAM [M]⁺= 399.14, 5′-dA [M+H]⁺= 252.11 and SAH [M+H]⁺= 385.13 are indicated. f, Schematic representation of the C38A-mutant and biochemical characterization. Middle panels: LC-MS analysis of the reaction before (blue trace) and after (red trace) 60 min incubation. SAM [M]⁺= 399.14, 5′-dA [M+H]⁺= 252.11 and SAH [M+H]⁺= 385.13 are indicated. Right panel: Time course analysis of the reaction. g, Schematic representation of the Y115A-mutant and biochemical characterization. Right and middle panels: LC-MS analysis of the reaction before (blue trace) and after (red trace) 1 hr incubation. SAM [M]⁺= 399.14, 5′-dA [M+H]⁺= 252.11 and SAH [M+H]⁺= 385.13 are indicated. h, Schematic representation of the Y115F-mutant and biochemical characterization. Activity of the Y115F mutant in the absence (Left panel) or the presence (middle panel) of the peptide substrate. Right panel: LC-MS analysis of the reaction after 1 hr. All reactions were performed under anaerobic and reducing conditions with reconstituted enzymes and analyzed by LC-MS. Methylated peptide [M+H]⁺= 1510.79 (●,◯), 5′-dA [M+H]⁺= 252.11 (■, ☐) and SAH [M+H]⁺= 385.13 (▲, Δ) are indicated. Experiments were performed in duplicate. i, Schematic representation and EPR analysis of Mmp10. Ox: Oxidized enzyme as purified, Red: Reduced enzyme after anaerobic reconstitution and incubation with sodium dithionite. SAM: Reduced enzyme after incubation with SAM. g-values are indicated on the panels. In all panels, the radical SAM cluster ligated by Tyr-115, Cys-15, Cys-19 & Cys-22, the iron-loop coordinated by Cys-35, Cys-38, Cys-45 and Cys-48 and the cobalamin cofactor are indicated.