Deciphering the <i>O</i> -Carbamoylation Modification Mechanism in Venturicidin A Biosynthesis

Herein, VtdB was identified as an essential <i>O</i>-carbamoyltransferase that catalyzes the conversion of venturicidin B (<b>3</b>) to venturicidin A (<b>1</b>). Notably, a 2.8 Å resolution crystal structure of the VtdB-substrate complex (VtdB/VTD-B) revealed that <b>3</b> occupies a specific hydrophobic pocket within the Kael-like domain, where His35 plays a critical deprotonation role during enzymatic catalysis. These biochemical and structural research provides the first mechanistic insight into d-olivose-glycosylated macrolide recognition by the carbamoyltransferase, enabling its future engineering.