Nonglycosidic C–O bond formation catalyzed by a bifunctional pseudoglycosyltransferase ValL

The C₇N antibiotic validamycin A is an antifungal agent widely used as a crop protectant. It comprises a validoxylamine A unit linked to a glucose moiety, which is formed through a nonglycosidic C–N bond connecting a valienol moiety and a validamine moiety, a reaction catalyzed by the pseudoglycosyltransferase ValL. In this study, we analyzed the chemical composition of validamycins in Streptomyces hygroscopicus var. jinggangensis TL01. A series of novel oxygen-bridged analogues, namely, validenomycin, validomycin, and 1,1′-bis-valienol, were identified in the culture supernatants, and their chemical structures were elucidated using a combination of one- and two-dimensional nuclear magnetic resonance and mass spectrometry. Gene disruption and complementation experiments revealed that valL is essential for the biosynthesis of these new oxygen-bridged analogues of validamycins. Biochemical assays further demonstrated that ValL catalyzed the C–O bond formation between GDP-valienol and valienol-7-phosphate, producing 1,1′-bis-valienol-7-phosphate, which was subsequently dephosphorylated by ValO and glycosylated by ValG to yield validenomycin. Collectively, our findings revealed the unique ability of ValL to catalyze nonglycosidic C–O coupling, potentially enabling the generation of various chemical scaffolds for C₇N family antibiotics.