Evolutionarily divergent DUF4465 domains have a common vitamin B ₁₂ ‐binding function

The DUF4465 family (DUF, domain of unknown function) contains more than 1000 members distributed across eight bacterial clades with species from diverse microenvironments including various gut microbiomes, hydrothermal vents, and soil. In the gut commensal Bacteroides thetaiotaomicron (B. theta), DUF4465 containing proteins act as high-affinity B₁₂-binding proteins that scavenge this cofactor to ensure bacterial survival. Such B₁₂ capture is essential for bacteria that have lost the ability to synthesize B₁₂ de novo. This raises the question of whether B₁₂-binding is ubiquitous across this family of proteins. Here, we show that B₁₂-binding is a recurrent function of eight distantly related members of the DUF4465 family. It is reasonable to conclude that B₁₂-binding is a common function of most DUF4465 proteins. These results establish DUF4465 as a structurally conserved family of augmented β-jellyroll B₁₂-binding proteins with widespread roles in microbial competition for this essential cofactor. Impact statement DUF4465 defines a widespread, structurally conserved bacterial cobalamin-binding domain and provides a promising scaffold for protein-based B₁₂ capture and purification.