Banasik, M. | Search bioRxiv

bioRxiv2025

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.

biochemistry↗

bioRxiv2025

Extracellular Vesicle-Linked Vitamin B12 Acquisition via Novel Binding Proteins in Bacteroides thetaiotaomicron

The acquisition and utilization of vitamin B12 (cobalamin) are essential for the metabolic functions of many gut bacteria, including Bacteroides thetaiotaomicron, which relies on external sources of cobalamin due to its inability to synthesize it de novo. To scavenge cobalamin efficiently, B. thetaiotaomicron employs a sophisticated cobamide uptake system comprising multiple operons encoding outer membrane binding proteins and transporters. This study identifies and characterizes several novel cobalamin-binding proteins and elucidates their roles in cobamide uptake and delivery via bacterial extracellular vesicles (BEVs), highlighting their siderophore-like function for competitive nutrient acquisition. We demonstrate that BtuJ1 and BtuJ2, members of the IPR027828 protein family, bind cobalamin with distinct structural and kinetic properties and are key components of BEVs responsible for cobamide capture and transfer to cells. Structural analyses reveal a conserved augmented {beta}-jellyroll architecture in these proteins, with tyrosine residues playing a central role in stabilizing cobalamin binding. Comparative proteomics of BEVs and cells under cobamide starvation underscore the selective enrichment of BtuJ proteins in BEVs, suggesting a specialized mechanism for nutrient acquisition. Additionally, we identify another novel B12-binding protein, BtuK1. We further establish BtuL as a critical player in early BEV release and propose a theoretical model in which BEVs function similarly to siderophores, scavenging cobalamin in the environment and delivering it to cells via specific receptors. This study provides new insights into the interplay between BEV-mediated transport, cobamide uptake, and the metabolic strategies employed by gut bacteria to thrive in nutrient-limited environments.

biochemistry↗