Evolutionarily divergent DUF4465 domains have a common vitamin B₁₂-binding function

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• The DUF4465 protein family comprises over 1000 members found in diverse bacterial species and environments.
• In *Bacteroides thetaiotaomicron*, DUF4465 proteins are essential for scavenging vitamin B12 (cobalamin), a vital cofactor for bacteria unable to synthesize it.
• The prevalence of B12-binding across the DUF4465 family remained largely uncharacterized.

Purpose of the Study:

• To investigate the functional conservation of B12-binding within the DUF4465 protein family.
• To determine if B12-binding is a ubiquitous or recurrent function across diverse DUF4465 members.
• To establish the structural and functional significance of DUF4465 proteins in microbial B12 acquisition.

Main Methods:

• Bioinformatic analysis of DUF4465 family distribution and sequence conservation.
• Experimental characterization of B12-binding activity in selected DUF4465 family members.
• Structural analysis of DUF4465 proteins to identify conserved B12-binding motifs.

Main Results:

• B12-binding was confirmed as a recurrent function in eight distantly related DUF4465 family members.
• The DUF4465 domain defines a structurally conserved, augmented β-jellyroll fold adept at binding cobalamin.
• These findings suggest B12-binding is a common functional trait for the majority of DUF4465 proteins.

Conclusions:

• DUF4465 proteins represent a widespread and structurally conserved family of bacterial B12-binding proteins.
• This family plays significant roles in microbial competition for the essential cofactor, vitamin B12.
• The DUF4465 domain serves as a promising scaffold for developing protein-based B12 capture and purification technologies.