Structural and Molecular Basis of the Catalytic Mechanism of Geranyl Pyrophosphate C6‐Methyltransferase: Creation of an Unprecedented Farnesyl Pyrophosphate C6‐Methyltransferase

Abstract

Prenyl pyrophosphate methyltransferases enhance the structural diversity of terpenoids.H owever,t he molecular basis of their catalytic mechanisms is poorly understood. In this study,u sing multiple strategies,w ec haracterized ag eranyl pyrophosphate (GPP) C6-methyltransferase,BezA. Biochemical analysis revealed that BezA requires Mg 2+ and solely methylates GPP.T he crystal structures of BezA and its complex with S-adenosyl homocysteine were solved at 2.10 and 2.56 ,r espectively.F urther analyses using site-directed mutagenesis,m olecular docking, molecular dynamics simulations,a nd quantum mechanics/molecular mechanics calculations revealed the molecular basis of the methylation reaction. Importantly,t he function of E170 as ac atalytic base to complete the methylation reaction was established. We also succeeded in switching the substrate specificity by introducing aW 210A substitution, resulting in an unprecedented farnesyl pyrophosphate C6-methyltransferase.

QM/MM Study of the Catalytic Mechanism and Substrate Specificity of the Aromatic Substrate C-Methyltransferase Fur6

“…The methylation follows the S N 2 mechanism, similar to the previous experimental and computational studies on other known methyltransferases. 19,26,27,51 The nucleophilic attack from the C3 atom on the methyl group of SAM produces SAH and an intermediate in which the C3 atom is methylated with a ΔE ‡ of approximately 8.6 kcal mol −1 . The methylated intermediate is then rearomatized through a water-mediated proton transfer pathway after it is presumably ejected into the water solvent.…”

“…Moreover, the C3 atom of PHN, methyl group, and sulfur atom of SAM stayed in an almost linear configuration (177.48°), which is consistent with the S N 2 reaction mechanism in other methyltransferases. 19,51 In the intermediate state (Figure 5C), the methyl group covalently bonded to the C3 atom of PHN, while SAM converted to SAH. The calculated ΔE ‡ of TS1 from the reactant state was 8.6 kcal mol −1 (Figure 5D), and the calculated ΔE of the intermediate state was −32.7 kcal mol −1 .…”

“…RI18. 19 we presented a model of the PHN-SAM-Fur6 ternary complex by using a docking simulation. However, when the active site of Fur6 is compared to that of CouO, the crucial residues His120 and Arg121 found in CouO are not conserved in Fur6.…”

“…Water molecules more than 5.0 Å from the protein atoms were removed to reduce the calculation cost. Since the B3LYP/6-31+G(d,p) 44,45 level of theory in the QM region and AMBER ff14SB force field/GAFF2 in the MM region have shown reliable accuracy and computational performance in our previous methyltransferase study, 19 we also chose these calculative levels for this study. The covalent boundary between the QM and MM regions was capped by hydrogenlinking atoms.…”

Enantioselectivity and Reactivity Enhancement by 1,1,3,3-Tetramethylguanidine in Bisguanidinium-Catalyzed Asymmetric Alkylation for Construction of Indole Alkaloid Marine Natural Products

“…Longchain geranyl is widely present in natural products with a variety of biological activities. 8 To our delight, the geranyl group was introduced to products 3k and 3l in high yields and enantioselectivities under the optimized conditions. Alkylation with phenyl-substituted allyl bromide showed high reactivity and provided 3m and 3n in high yields and enantioselectivities.…”