BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the β-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the β-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (K_D = 4.0 μM), followed by the formation of a covalent bond (rate constant of 0.163 s⁻¹), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.

BtuM 是一种细菌钴胺素转运蛋白，在碱基关闭状态下与转运的底物结合，半胱氨酸残基通过硫钴键提供中心钴离子的 α 轴配位。结合导致以氰基作为 β 轴配体的钴胺素变体脱氰化。在这里，我们报告了与两种钴胺素变体（羟钴胺素和氰钴胺素）结合的未标记 BtuM 的晶体结构，并揭示了负责 β 轴配位的天然残基 His28。这种协调此前曾被 His 标记的 BtuM 的非天然组氨酸所掩盖。 BtuM 最初以低亲和力 (K _D  = 4.0 μM) 可逆地结合钴酰胺，随后形成共价键（速率常数为 0.163 s ^-1 ），该模型符合底物结合和钴胺素脱氰化的动力学数据BtuM 的前体钴酰胺。共价结合模式表明了任何其他转运蛋白都没有使用的机制。