Details

Autor(en) / Beteiligte

• Martínez Felices, Jose M.
• Barreto, Yan Borges
• Thangaratnarajah, Chancievan
• Whittaker, Jacob J.
• Alencar, Adriano M.
• Guskov, Albert
• Slotboom, Dirk J.

Titel

Cobalamin decyanation by the membrane transporter BtuM

Ist Teil von

• Structure (London), 2024-08, Vol.32 (8), p.1165-1173.e3

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2024

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 (KD = 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. [Display omitted] •Cobalamin transporter BtuM structures reveal His28 as native β-axial coordinate•Experiments and kinetic modeling reveal a two-step binding mechanism•Weak and reversible initial substrate binding is followed by covalent trapping•Substrate remains covalently bound after binding, obscuring the transport mechanism Martínez Felices et al. reveal the structural basis of the covalent association between BtuM, a bacterial transporter, and its substrate cobalamin (vitamin B12). The binding kinetics, in which the substrate is chemically altered, was elucidated using spectrometric techniques and a mathematical modeling approach.