2.A.88 The Vitamin Uptake Transporter (VUT or ECF) Family
BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Comparative genomic and experimental analyses (Rodionov et al., 2006) revealed the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids and vitamins. These systems resemble ATP-binding cassette (ABC; TC #3.A.1.25.1)-containing transporters and contain typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one third of the widespread bioY genes is linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis or are unlinked to either biotin synthesis or other biotin transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the α-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant E. coli cells. Kinetic analyses identified BioY as a high-capacity transporter which was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue of BioM demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that the BioM, N, and Y proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results suggest a mechanistically novel group of membrane transporters.
Some transporters have a conserved transmembrane protein and two nucleotide binding proteins similar to those of ABC transporters. However, unlike typical ABC transporters (E.I. Sun & M.H. Saier, unpublished results), they use small integral membrane proteins that are postulated to capture specific substrates. Our studies have shown that both of the integral membrane protein constituents of these systems are distantly related but homologous, and in this respect they resemble typical ABC porters. We postulate that these two transmembrane proteins comprise the pathway for transmembrane transport.
Rodionov et al., 2009 identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) examined by Rodionov et al., 2009 have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. Rodionov et al., 2009 proposed the name energy-coupling factor transporters for the new class of putative ABC membrane transporters. These membrane proteins are homologues to ABC-2 exporters. When evidence is minimal for association with an ABC-type ATP-hydrolyzing subunit, these porters are placed in category 2.A (secondary carriers; e.g., 2.A.88).
The uptake porters of the ABC superfamily and of the vitamin/small molecule transporters described by Rodionov et al., 2009 are homologous to the porters in the VUT family (2.A.88). In fact, our studies indicated that all uptake porters of the ABC superfamily are of the ABC2 type. When evidence suggests that homologous membrane transport proteins of the ABC2 type couple transport to ATP hydrolysis using a homologue of the ABC-type ATPases, we list these proteins in the ABC superfamily. If there is no such evidence, (e.g., experimental evidence and the occurrence of the gene for the membrane transporter protein is in an operon that lacks the ATPase and auxillary subunit) then the porter is placed into family 2.A.88.
Erkens et al. (2011) presented the crystal structure of the thiamine-specific S-component of the ECF-type ABC transporter, ThiT from Lactococcus lactis at 2.0 Å. Extensive protein-substrate interactions explain its high binding affinity for thiamine (Kd ~ 10-10 M). ThiT has a fold similar to that of the riboflavin-specific S-component RibU, with which it shares only 14% sequence identity. Two alanines in a conserved motif (AxxxA) located on the membrane-embedded surface of the S-components mediate the interaction with the energizing module. A general transport mechanism for ECF transporters is proposed (Erkens et al., 2011).
ATP binding to the ATPase, EcfAA', drives a conformational change that dissociates the EcfS subunit from the EcfAA'T module. Upon release, the RibU S subunit then binds the riboflavin transport substrate, and S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Thus, ECF transporters capture the transport substrate and reproduce in vivo observations on S-subunit competition (Karpowich et al. 2015).
The reaction catalyzed by BioY is:
biotin (out) → biotin (in).
The reaction catalyzed by BioMNY is:
biotin (out) + ATP → biotin (in) + ADP + Pi