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.