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

Domains of Unknown Function (DUFs) comprise a large portion of the bacterial proteome, yet their biological roles remain poorly understood. We recently identified two DUF4465 proteins (IPR027828 family proteins), BtuJ1 and BtuJ2, in the vitamin B ₁₂ –auxotrophic gut commensal Bacteroides thetaiotaomicron , which act as high-affinity B ₁₂ –binding proteins that scavenge the cofactor to ensure survival. Such B ₁₂ capture is essential for bacteria that have lost the ability to synthesize B ₁₂ de novo . The DUF4465 family contains more than 1,000 members distributed across eight bacterial clades in gut microbiome and marine environments, raising the question of whether B ₁₂ -binding is ubiquitous across this family. Here, we show that B ₁₂ -binding is conserved across five additional sequence-diverse DUF4465 proteins bringing the total we have characterized to seven. Structural and biochemical analyses, including the crystal structure of D5EK51 from Coraliomargarita akajimensis bound to B ₁₂ , reveal a conserved augmented β-jellyroll fold and a shared B ₁₂ -binding motif. Together, these findings establish DUF4465 as a structurally conserved family of B ₁₂ -binding proteins and point to their widespread role in microbial competition for this essential cofactor.