Cobalamin decyanation by the membrane transporter BtuM

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• BtuM is a bacterial transporter essential for cobalamin (vitamin B12) uptake.
• Previous studies utilized His-tagged BtuM, potentially obscuring native substrate binding mechanisms.
• Cobalamin transport involves specific axial coordination of the central cobalt ion.

Purpose of the Study:

• To determine the crystal structures of untagged BtuM bound to hydroxycobalamin and cyanocobalamin.
• To identify the native residue responsible for β-axial coordination in BtuM.
• To elucidate the kinetic mechanism of cobinamide binding and decyanation by BtuM.

Main Methods:

• X-ray crystallography of untagged BtuM complexed with cobalamin variants.
• Kinetic analysis of cobinamide binding and decyanation.
• Structural and mechanistic modeling of BtuM-cobalamin interactions.

Main Results:

• Crystal structures reveal His28 as the native residue for β-axial coordination, previously masked by His tags.
• BtuM binds cobinamide with initial reversible low-affinity interaction (K_D = 4.0 μM).
• A subsequent covalent bond formation (rate constant = 0.163 s^-1) accompanies substrate decyanation.

Conclusions:

• Untagged BtuM structures elucidate the native binding site and His28's role in β-axial coordination.
• BtuM employs a unique two-step binding mechanism involving reversible and covalent interactions.
• This covalent binding mode represents a novel mechanism for substrate transport not observed in other transport proteins.