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 However, unlike typical ABC transporters (E.I. Sun & M.H. Saier, unpublished results), they use small integral membrane proteins that are postulated to capture specific substrates. Both of the integral membrane protein constituents of these systems may be distantly related, and in this respect they resemble typical ABC porters. Possibly, these two transmembrane proteins comprise the pathway for transmembrane transport. However, the VUT family member, TrpP of Bacillus subtilis (2.A.88.4.1) and the ThiW ABC membrane protein homologue, 3.A.1.26.2, are clearly related by common descent. Families 2.A.88 and 2.A.87 which are part of a superfamily, and 3.A.1.26, are homologous but function as secondary versus primary active transporters, respectively. Only the S subunit is required for transport as a secondary porter.

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



This family belongs to the .

 

References:

Ames, T.D., D.A. Rodionov, Z. Weinberg, and R.R. Breaker. (2010). A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate. Chem Biol 17: 681-685.

Erkens, G.B. and D.J. Slotboom. (2010). Biochemical characterization of ThiT from Lactococcus lactis: a thiamin transporter with picomolar substrate binding affinity. Biochemistry 49: 3203-3212.

Erkens, G.B., R.P. Berntsson, F. Fulyani, M. Majsnerowska, A. Vujičić-Žagar, J. Ter Beek, B. Poolman, and D.J. Slotboom. (2011). The structural basis of modularity in ECF-type ABC transporters. Nat Struct Mol Biol 18: 755-760.

Finkenwirth F., Kirsch F. and Eitinger T. (2013). Solitary BioY proteins mediate biotin transport into recombinant Escherichia coli. J Bacteriol. 195(18):4105-11.

Fisher, D.J., R.E. Fernández, N.E. Adams, and A.T. Maurelli. (2012). Uptake of biotin by Chlamydia Spp. through the use of a bacterial transporter (BioY) and a host-cell transporter (SMVT). PLoS One 7: e46052.

Hebbeln, P., D.A. Rodionov, A. Alfandega, and T. Eitinger. (2007). Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module. Proc. Natl. Acad. Sci. USA 104: 2909-2914.

Karpowich NK., Song JM., Cocco N. and Wang DN. (2015). ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat Struct Mol Biol. 22(7):565-71.

Kirsch F., Frielingsdorf S., Pohlmann A., Ziomkowska J., Herrmann A. and Eitinger T. (2012). Essential amino acid residues of BioY reveal that dimers are the functional S unit of the Rhodobacter capsulatus biotin transporter. J Bacteriol. 194(17):4505-12.

Majsnerowska M., Hanelt I., Wunnicke D., Schafer LV., Steinhoff HJ. and Slotboom DJ. (2013). Substrate-induced conformational changes in the S-component ThiT from an energy coupling factor transporter. Structure. 21(5):861-7.

Ravcheev, D.A., M.S. Gel'fand, A.A. Mironov, and A.B. Rakhmaninova. (2002). [Purine regulon of γ-proteobacteria: a detailed description]. Genetika 38: 1203-1214.

Rodionov, D.A., A.G. Vitreschak, A.A. Mironov, and M.S. Gelfand. (2002). Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J. Biol. Chem. 277: 48949-48959.

Rodionov, D.A., A.G. Vitreschak, A.A. Mironov, and M.S. Gelfand. (2003). Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J. Biol. Chem. 278: 41148-41159.

Rodionov, D.A., P. Hebbeln, A. Eudes, J. ter Beek, I.A. Rodionova, G.B. Erkens, D.J. Slotboom, M.S. Gelfand, A.L. Osterman, A.D. Hanson, and T. Eitinger. (2009). A novel class of modular transporters for vitamins in prokaryotes. J. Bacteriol. 191: 42-51.

Rodionov, D.A., P. Hebbeln, M.S. Gelfand, and T. Eitinger. (2006). Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters. J. Bacteriol. 188: 317-327.

Sarsero, J.P., E. Merino, and C. Yanofsky. (2000). A Bacillus subtilis gene of previously unknown function, yhaG, is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport. J. Bacteriol. 182: 2329-2331.

Zallot, R., Y. Yuan, and V. de Crécy-Lagard. (2017). The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ₀) Transport. Biomolecules 7:.

Examples:

TC#NameOrganismal TypeExample
2.A.88.1.1

Biotin transporter, BioY (Biotin ECF transporter S component) (Hebbeln et al., 2007; Rodionov et al., 2009).

Bacteria

BioY of Rhizobium etli (Q6GUB0)

 
2.A.88.1.2The BioY homologue (190 aas; 6TMSs)BacteriaBioY homologue of Rickettsia typhi (Q68X47)
 
2.A.88.1.3

Biotin transporter, BioY (Biotin ECF transporter S component) (Hebbeln et al. 2007; Rodionov et al., 2009). The functional unit is a dimer (Kirsch et al., 2012).

Bacteria

BioY of Rhodobacter capsulatus (D5ARG8)

 
2.A.88.1.4

Biotin transporter, BioY of 201 aas and 6 TMSs.  Transports biotin with high affinity without other subunits (Finkenwirth et al. 2013).

Proteobacteria

BioY of Oceanicola batsensis

 
2.A.88.1.5

Biotin transporter, BioY of 192 aas and 5 TMSs.  Transports biotin without other subunits (Finkenwirth et al. 2013).

Proteobacteria

BioY of Rhodopseudomonas palustris

 
2.A.88.1.6

BioY of 195 aas and 6 TMSs.  Transports biotin with high affinity without additional subunits (Finkenwirth et al. 2013).

Proteobacteria

BioY of Ruegeria pomeroyi (Silicibacter pomeroyi)

 
2.A.88.1.7

BioY biotin uptake porter.  (note: no AT energizer was found encoded in the genome of C. trachomatis (Fisher et al. 2012).

Chlamydiae

BioY of Chlamydia trachomatis

 
2.A.88.1.8The distant BioY homologue (183 aas; 6 TMSs)BacteriaBioY homologue of Bordetella avium (Q2KUS5)
 
2.A.88.1.9

Biotin transporter, BioY (Biotin ECF transporter S component) (Hebbeln et al., 2007; Rodionov et al., 2009).

Bacteria

BioY of Bacillus subtilis (O07620)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.10.1


Putative thiazole transporter, ThiW. (thiazole ECF transporter S component) Regulated by a TPP riboswitch (Rodionov et al. 2009).

Firmicutes

ThiW of Streptococcus pneumoniae (Q97RS0)

 
Examples:

TC#NameOrganismal TypeExample
Examples:

TC#NameOrganismal TypeExample
Examples:

TC#NameOrganismal TypeExample
2.A.88.2.1

Folate transporter, FolT (Folate ECF transporter S component) (Rodionov et al., 2009). Regulated by a THF riboswitch (Ames et al. 2010).

 

Firmicutes

FolT of Clostridium acetobutylicum (Q97GE9)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.3.1

Thiamin transporter, ThiT (Thiamin ECF transporter S component) (Rodionov et al. 2002; Rodionov et al. 2009).

Bacteria

YuaJ of Bacillus subtilis (O32074)

 
2.A.88.3.2

Thiamin transporter, ThiT (Thiamin ECF transporter S component) (Rodionov et al. 2002; Rodionov et al., 2009). High affinity thiamin transporter ThiT (Ka=120 pM). Other substrates include TPP, TMP and pyrithiamin with nM binding constants at 1:1 stoichiometry (protein:ligand). Binding depends on a tryptophan-rich loop between TMSs 5 and 6 (Erkens and Slotboom, 2010). 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 has been proposed (Erkens et al., 2011).  Substrate binding induces conformational changes in ThiT (Majsnerowska et al. 2013).

Firmicutes

ThiT of Lactococcus lactis (A2RI47)

 
2.A.88.3.3

Thiamin transporter, ThiT (Thiamin ECF transporter S component) (Rodionov et al. 2002; Rodionov et al. 2009).

 

 

Firmicutes

ThiT of Lactobacillus casei (Q037U3)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.4.1

Tryptophan transporter TrpP (YhaG; Tryptophan ECF transporter S component) (Rodionov et al., 2009; Sarsero et al., 2000)

Firmicutes

TrpP of Bacillus subtilis (O07515)

 
2.A.88.4.2

Putative dimethylbenzimidazole porter, CblT (dimethylbenzimidazole ECF transporter S component) Rodionov et al., 2009) Regulated by a Vitamin B12 riboswitch (Rodionov et al. 2003).

Firmicutes

CblT of Clostridium botulinum (A5I0E4)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.5.1

Putative niacin uptake transporter, NiaX (niacin ECF transporter S component) (Rodionov et al., 2009). 

Firmicutes

NiaX of Streptococcus pyogenes (Q99Z31)

 
2.A.88.5.2

Putative niacin uptake transporter, NiaX (niacin ECF transporter S component) Rodionov et al., 2009).

Bacteria

NiaX of Lactococcus lactis subsp. cremoris

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.6.1

Putative queuosine precursor uptake transporter, QrtT  (queosine ECF transporter S component) (Rodionov et al., 2009) (5 or 6 TM5s).

Firmicutes

QrtT of Lactobacillus sakei (Q38XE8)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.7.1

Putative lipoate transporter, LipT (lipoate ECF transporter S component) (Rodionov et al., 2009).

Mollicutes

LipT of Onion yellows phytoplasma (Q6YQR5)

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.8.1

Putative queuosine precursor transporter, YpdP (queuosine ECF transporter S component). Regulated by a preQ1 riboswitch (243 aas, 7 TMSs).

Firmicutes

YpdP of Staphylococcus lugdunensis (D3QD12)

 
2.A.88.8.2

YhhQ transporter (Duf165)

Bacteria

YhhQ transporter of Bacillus subtilis (P54163)

 
2.A.88.8.3

YpdP transporter (Duf165) (229 aas; 7 TMSs)

Bacteria

YpdP of Bacillus subtilis (G4P330)

 
2.A.88.8.4

Duf165 transporter (229 aas; 5 TMSs)

Archaea

Transporter of Methanosarcina mazei (Q8PW04)

 
2.A.88.8.5

Putative ACR family transporter (DUF165) (261 aas; 6 TMSs)

Bacteria

ACR family transporter of Stenotrophomonas maltophilia (B2FPS5)

 
2.A.88.8.6

YhhQ protein (DUF165) (encoded within the purine regulon (PurR) (Ravcheev et al., 2002)) (221 aas; 6 TMSs).  It may be a 7-cyano-7-deazaguanine (preQ₀) transporter. PreQ0 is the product of the reaction catalyzed by GTP cyclhydrolase I, important for the synthesis of folic acid, and an intermediate of interest due to its central role in tRNA and DNA modification and secondary metabolism (Zallot et al. 2017).

Bacteria

YhhQ of E. coli (B3WJF3)

 
2.A.88.8.7

Uncharacterized protein; YpdP homologue of 248 aas and 6 TMSs.

Spirochaetes

UP of Treponema succinifaciens

 
Examples:

TC#NameOrganismal TypeExample
2.A.88.9.1

Predicted queuosine precursor transporter, QueT (queuosine ECF transporter S component) (Rodionov et al., 2009) (169 aas; 5 TMss).

Firmicutes

QueT of Lactococcus lactis (A2RM05) 

 
2.A.88.9.2

Predicted queuosine precursor transporter, QueT (queuosine ECF transporter S component) (Rodionov et al., 2009) (187 aas; 4 TMSs).

Firmicutes

QueT of Leuconostoc gasicomitatum (D8MFQ0)

 
2.A.88.9.3

Uncharacterized protein of 378 aas and 6 TMSs, one in the middle of the N-terminal hydrophilic domain of about 220 aas, and the remaining 5 together in the C-terminal domain. The N-terminal domain is the QueC or ExsB family, where the former is involved in queosine biosynthesis and may be a regulator, while the latter is the trannsmembrane transport protein. This is the only protein in the NCBI protein database with this fusion, so it may be an artifact.

UP of Candidatus Korarchaeota archaeon (hot springs metagenome)