Evolutionarily divergent DUF4465 domains have a common vitamin B12-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 B12-auxotrophic gut commensal Bacteroides thetaiotaomicron, which act as high-affinity B12-binding proteins that scavenge the cofactor to ensure survival. Such B12 capture is essential for bacteria that have lost the ability to synthesize B12 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 B12-binding is ubiquitous across this family. Here, we show that B12-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 B12, reveal a conserved augmented {beta}-jellyroll fold and a shared B12-binding motif. Together, these findings establish DUF4465 as a structurally conserved family of B12-binding proteins and point to their widespread role in microbial competition for this essential cofactor.