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 <i>C</i> <i>-</i> <i>N</i> 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 <i>Streptomyces hygroscopicus</i> var. <i>jinggangensis</i> 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 <i>valL</i> is essential for the biosynthesis of these new oxygen-bridged analogues of validamycins. Biochemical assays further demonstrated that ValL catalyzed the <i>C-O</i> 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 <i>C-O</i> coupling, potentially enabling the generation of various chemical scaffolds for C₇N family antibiotics.