Abstract
Pterin-containing natural products have diverse functions in life, but an efficient and easy scheme for their in vitro synthesis is not available. Here we report a chemoenzymatic 14-step, one-pot synthesis that can be used to generate 13C- and 15N-labeled dihydrofolates (H2F) from glucose, guanine, and p-aminobenzoyl-l-glutamic acid. This synthesis stands out from previous approaches to produce H2F in that the average yield of each step is >91% and it requires only a single purification step. The use of a one-pot reaction allowed us to overcome potential problems with individual steps during the synthesis. The availability of labeled dihydrofolates allowed the measurement of heavy-atom isotope effects for the reaction catalyzed by the drug target dihydrofolate reductase and established that protonation at N5 of H2F and hydride transfer to C6 occur in a stepwise mechanism. This chemoenzymatic pterin synthesis can be applied to the efficient production of other folates and a range of other natural compounds with applications in nutritional, medical, and cell-biological research.
Highlights
Pterin is a common motif found in natural products
The biosynthetic pathway to folate in E. coli uses the building blocks D-glucose, guanine, and p-aminobenzoyl-L-glutamate
D-Glucose serves as the starting material, which is transformed into phosphoribose pyrophosphate (PRPP) in five steps that are catalyzed by hexokinase (HK), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), phosphoriboisomerase (PRI), and ribose-phosphate pyrophosphokinase.[44]
Summary
Pterin is a common motif found in natural products. Folate, the essential vitamin that fuels the one-carbon cycle for the biosynthesis of nucleotide and amino acid building blocks, was one of the first natural products found to contain pterin.[1,2] The metabolic importance of pterins is illustrated by their integration into enzyme cofactors such as molybdopterin and tetrahydrobiopterin.[3−5] Pterin natural products are used as pigments in the butterflies Catopsilia argante and Appias nero,[6] whereas biopterin-α-glucoside serves as a natural sunscreen that protects cellular contents from photoinduced damage in photosynthetic cyanobacteria.[7]. If the protonation and hydride transfer steps were concerted, 15N- and 13C-labeled H2Fs should both yield measurable KIEs;[19,21,22] this interdependence may lead to an additive effect in the multiple heavy-atom isotope effect measurement with the double-labeled substrate.[19,21,22] In other words, our results suggest that the pre-steady-state kinetic measurement at pH 7.0 reveals only the step of hydride transfer because protonation of N5 is in rapid equilibrium and the ensemble of reaction-ready conformations is mostly populated with protonated H2F These results confirm the validity of previous solvent KIE and site-directed mutagenesis studies, which concluded that the sequence of chemical events (protonation and hydride transfer) is distinct and strictly ordered.[64,68] Overall, the results provided here strongly support a mechanism where protonation and hydride transfer are independent of each other and occur in a stepwise fashion
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