Reinvestigation of the C-Glycoside Synthase in Alnumycin Biosynthesis Reveals a Conserved Mechanism of C–C Bond Formation

C-Nucleosides are defined by their unusual C–C glycosidic linkage between the nucleobase and the monosaccharide moiety, which distinguishes them from common N-nucleosides. Several enzymes have been identified to catalyze this atypical C–C bond formation. For instance, YeiN catalyzes the reversible cleavage of pseudouridine 5′-phosphate, yielding ribose 5-phosphate (R5P) and uracil via a Schiff base intermediate formed between R5P and an active-site lysine residue. In alnumycin biosynthesis, the C–C glycosidic bond between R5P and a naphthoquinone heterocycle, prealnumycin, has been shown to be installed by AlnA. While AlnA shares 41% sequence identity with YeiN, a distinct mechanism involving an ene-diol tautomer of R5P had been proposed based on previous biochemical studies and X-ray crystallography. Herein, the mechanism of AlnA is reevaluated using juglone (5-hydroxy-1,4-naphthalenedione) as a prealnumycin analog. By employing isotopologues and protein mass spectrometry, the involvement of an ene-diol intermediate and an alternative Morita–Baylis–Hillman mechanism in AlnA catalysis can both be ruled out. Further analysis of juglone reactivity showed that it can be reduced either enzymatically when coupled to glucose oxidase or nonenzymatically through autoreduction yielding 1,4,5-naphthalenetriol. This hydroquinone derivative of juglone serves as the true substrate of AlnA such that the C-glycosylation mechanism is no different from that of YeiN. These findings unravel the correct substrate of the C-glycoside synthase AlnA and unify the mechanisms of AlnA, YeiN, and other C-glycoside synthases. These results highlight that accurate substrate identification is essential for mechanistic study of enzyme catalysis and call for a reevaluation of the biosynthetic pathway of alnumycin and other naphthoquinone-derived natural products.