Enzymatic Conversion from a Vicinal Diol to a Vicinal Diketone: Computational Study of the Conserved 6-Pyruvoyltetrahydropterin-Forming Stage in Mammalian and Bacterial PTPS

As a zinc- and magnesium-dependent triphosphatase, 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyzes the conversion of dihydroneopterin triphosphate (H2NTP) to 6-pyruvoyltetrahydropterin (PPH4) and a triphosphate ion, representing a typical reaction for vicinal diketone biosynthesis from vicinal diol. The molecular mechanism of PTPS remains elusive owing to the lack of structural data containing native substrate H2NTP and magnesium ion. Here, we performed a quantum mechanics/molecular mechanics (QM/MM) investigation based on a predicted PTPS structure, elucidating the complete atomistic reaction mechanism for triphosphate ion formation and stereoselective product generation. The overall catalytic reaction of PTPS proceeds via two half-reactions, each comprising four sequential proton-transfer steps. Both half-reactions involve the conversion of a hydroxyl group on the substrate into a carbonyl group, ultimately furnishing the pyruvoyl-containing product PPH4. The third proton transfer in the first half-reaction occurs preferentially on the Si-face of the critical carbanion intermediate, yielding the (R)-configured product, in agreement with the reported experimental detection results. These calculations not only unveil the structural origins of stereoselective vicinal diketone product formation, but also uncover a non-canonical triphosphate elimination pathway driven by a charge-center shift that facilitates C–O bond cleavage. These findings broaden the mechanistic paradigm of triphosphatase-mediated phosphate chemistry and offer foundational insights for synthesizing vicinal diketones from vicinal diols.