Abstract
Vitamin B12 (cobalamin) is a high-value yet scarce cofactor required for various metabolic processes, making its efficient handling important for maintaining metabolic homeostasis. While the involvement of ATP:cob(I)alamin adenosyltransferases (MMAB) in the synthesis, delivery, and repair of 5'-deoxyadenosylcobalamin (AdoCbl) is well established, the kinetic mechanisms that regulate this process, particularly its negative cooperativity, remain poorly understood. Understanding these mechanisms is key to clarifying how MMAB efficiently uses AdoCbl, prevents resource wastage, and supports bacterial survival in nutrient-limited environments. Using single-molecule relative fluorescence (SRF) spectroscopy, we found that conformation-gated binding is the driving force behind MMAB's preference for AdoCbl over hydroxocobalamin and is the underlying mechanism for negative cooperativity. This mechanism significantly slows down the binding of the second equivalent of AdoCbl, favoring the singly bound state. Our findings indicate that MMAB predominantly binds a single AdoCbl, optimizing the AdoCbl loading to methylmalonyl-CoA mutase. Additionally, our SRF approach also serves as a tool to explore other cofactor interactions, such as those between riboswitches and cobalamin derivatives, to provide insights into regulatory mechanisms of cobalamin sensing and gene regulation, which are crucial for bacterial adaptation to changing nutrient conditions
Competing Interest Statement
The authors have declared no competing interest.
