Structural and Mechanistic Insights into Dual Deprotonation by an Engineered GPP Methyltransferase: Discovery of Methyltransferases Generating Noncanonical Prenyl Diphosphates

The discovery and engineering of prenyl diphosphate C-methyltransferases have facilitated the understanding and expansion of terpenoid diversity. Here, we report the structural and mechanistic characterization of a methyltransferase variant HumMT W197G that produces unconventional C11 prenyl diphosphates through an unusual dual deprotonation mechanism. Through protein engineering, we expanded the substrate-binding pocket of Micromonospora humi methyltransferase (HumMT) by W197G substitution, converting the substrate specificity from DMAPP and IPP (C5) to GPP (C10). Notably, HumMT W197G exhibits both C4 and C2 deprotonation, producing both iso-2-methyl-GPP and 2-methyl-GPP. Using cryogenic electron microscopy (cryo-EM), we solved the structures of apo-HumMT W197G, HumMT W197G-SFG, and HumMT W197G-SFG-Mg2+-GPP complexes to resolutions of 3.14, 2.97, and 2.3 Å, respectively. Through structure-guided engineering, molecular docking, and molecular dynamics simulations, we identified E168 as a catalytic base facilitating deprotonation at both the C2 and C4 positions. Additionally, we discovered W197Y as a critical mutation that discriminates between IPP and DMAPP substrates, leading to the identification of a natural methyltransferase from Micromonospora echinospora that produces noncanonical C7 prenyl diphosphates 2,2-dimethyl-IPP and 2,4-dimethyl-DMAPP. Our findings expand the understanding of methyltransferase catalytic mechanisms and demonstrate successful structure-based protein engineering to modulate carbocation intermediate deprotonation, providing implications for expanding terpenoid diversity.