Unusual O–H Activation-Initiated C–C Bond Cleavage Reaction by a Nonheme Fe Enzyme in Antifungal Nucleoside Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent enzymes catalyze diverse reactions, generally initiated by Fe^IV=O mediated cleavage of C-H bonds with bond dissociation energies (BDE) of up to ∼100 kcal/mol. Here, we report the discovery of a novel reaction initiated by a significantly more challenging O-H bond cleavage (>100 kcal/mol). This activity was identified in PolD, an enzyme that regulates the sugar size in antifungal nucleoside biosynthesis by catalyzing the transformation of a bicyclic eight-carbon sugar substrate, 5'-amino-6'-hydroxy-octosyl acid 2'-phosphate (AHOAP), into a monocyclic six-carbon product, aminohexuronic acid 2'-phosphate (AHAP). Our studies demonstrate that PolD catalyzes a two-step reaction, in which AHOAP is first oxidized to 5'-amino-6'-keto-octosyl acid 2'-phosphate (AKOAP) via typical C-H activation, followed by a unique C-C bond cleavage on AKOAP to AHAP initiated by O-H activation. X-ray crystal structures of PolD and its homologue, PasI, the latter solved in complex with AHOAP, succinate, and vanadyl, a structural mimic of the Fe^IV-oxo intermediate, reveal a substrate binding mode that is consistent with both C-H and O-H homolysis. A comparison of the three enzymes, PasI, PolD, and MalI, all of which exhibit distinct C-C bond cleavage activities, suggests that precise substrate positioning to bring the target OH group of AKOAP close to the Fe^IV-oxo intermediate is critical for hydrogen atom transfer from this functional group. These results indicate a novel reactivity of the Fe^IV═O intermediate in Fe/α-KG enzymes, thereby expanding the reaction scope of this enzyme superfamily. The results also reveal the molecular mechanism of the divergent biosynthesis of antifungal nucleosides.