A molecular basis for stoichiometric enzyme encapsulation in the vitamin B2 biosynthesis compartment.

Encapsulating metabolic enzymes within protein cages enhances catalytic efficiency through substrate channeling. The vitamin B2 biosynthesis system, in which a dodecahedral lumazine synthase (LS) cage encapsulates a homotrimeric riboflavin synthase (RS), exemplifies this strategy, yet the molecular basis for this stoichiometric enzyme encapsulation has remained elusive. Here, cryogenic electron microscopy structures reveal a hierarchical assembly mechanism that ensures the defined host-guest ratio. RS C-terminal cage-localization signal peptides anchor at LS pentamer-pentamer interfaces early during assembly, stabilizing open intermediates that, together with delayed later-stage cage closure, extend the loading window until guest incorporation is complete. RS spatial occupancy avoids overloading, while a molecular lock upon final closure prevents disassembly. The elucidated anchoring mechanism enabled structure-based phylogenetic analysis across diverse organisms, suggesting multiple independent evolutionary origins of this modular encapsulation strategy. This naturally occurring architecture provides design principles for engineering synthetic catalytic compartments with programmable stoichiometric control.