Catalytic amino acid residues of FCE. (A) Under standard conditions (see Materials and Methods), 2.5 nM of FCE WT enzyme converted ≥90% of ppp-RNA into pp-RNA in the absence of GTP and SAM (Reaction 1), ∼90% pp− into Gppp− in the presence of GTP (Reaction 2) and ∼80% of Gppp− into m7Gppp− in the presence of SAM (Reaction 3). (B) Mutations of the Glu residues of the ELE motifs knocked out TPase activity but did not affect GTase or N7MTase activities. Mutants E26A/E28A, E184A/E186A, and E26A/E28A/E184A/E186A exhibited the same trend. (C) Mutation of Lys282 of the conserved KTDG motif to Met eliminated the formation of the GMP-GTase covalent intermediate (left panel). The mutant K282M exhibited undetectable GTase activity (Reaction 2) but exhibited high level of TPase (Reaction 1) and N7MTase activity (Reaction 3). (D) Mutation of Tyr712 in the conserved SAM-binding motif to Ala significantly decreased the N7MTase activity (Reaction 3) compared to the WT FCE enzyme but not TPase (Reaction 1) or GTase activity (Reaction 2).
