Conformation-gated binding underlies kinetic asymmetry and negative cooperativity in ATP:cob(I)alamin adenosyltransferase.

Vitamin B₁₂ (cobalamin) is a high-value yet scarce cofactor critical for metabolic homeostasis, necessitating efficient handling mechanisms. ATP:cob(I)alamin adenosyltransferase (MMAB) plays a central role in synthesizing, delivering, and repairing 5'-deoxyadenosylcobalamin (AdoCbl), but the kinetic mechanisms regulating this process, including negative cooperativity, remain unclear. Using single-molecule relative fluorescence spectroscopy, we reveal that conformation-gated binding mechanism, involving a required structural rearrangement prior to the first cofactor association, dictates MMAB's interaction kinetics. This mechanism slows the association of a second AdoCbl, resulting in strong negative cooperativity, favoring the singly bound state, and optimizing AdoCbl handling. This gating mechanism, supported by direct observation of a kinetic intermediate, also contributes to MMAB's preferential handling of AdoCbl over hydroxocobalamin, highlighting MMAB's effective cofactor utilization, supporting bacterial survival in nutrient-limited environments. Furthermore, our approach offers a platform to study cofactor interactions, including cobalamin sensing and gene regulation, shedding light on bacterial adaptation to nutrient fluctuations.