TCDB is operated by the Saier Lab Bioinformatics Group

1.A.16 The Formate-Nitrite Transporter (FNT) Family

FNT family members have been sequenced from Gram-negative and Gram-positive bacteria, archaea and yeast. The prokaryotic proteins of the FNT family probably function in the transport of the structurally related compounds, formate, nitrite and hydrosulfide. Formate, nitrite and hydrosulphide transporters respectively are clustered into two (FocA and FdhC), three (NirC-α, NirC-β and NirC-γ) and one (HSC) subfamilies, plus two (YfdC-α and Yfc-β) of unknown specificity (Mukherjee et al. 2017; see below). Certain positions in the two constriction regions and some residues facing the interior show subfamily-specific conservation.  Anions, as such, seem not to traverse the FNT pore (Atkovska and Hub 2017). Instead, anion binding in the pore is energetically coupled to protonation of a centrally located histidine.The histidine can protonate the permeating anion, thereby enabling its release. Such a mechanism may facilitate both export and import of substrates, with or without proton co-transport (Atkovska and Hub 2017).

FNTs exhibit dual transport functionality: at neutral pH, electrogenic anion currents are detectable, whereas upon acidification, transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of energy metabolism, such as l-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. The proton relay postulate suggests proton transfer from a highly conserved histidine centrally positioned in the transport path, but the dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Helmstetter et al. 2019 defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also showed that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including l-lactate. A low substrate affinity is typical for FNTs, and,  proton motive force-dependent transport is observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules (Helmstetter et al. 2019).

With the exception of the yeast protein (627 amino acyl residues), all members of the family are of about 250-300 residues in length and exhibit 6-8 putative transmembrane α-helical spanners (TMSs). In one case, that of the E. coli FocA protein, a 6 TMS topology has been established. The yeast protein has a similar apparent topology but has a large C-terminal hydrophilic extension of about 400 residues. Formate export and import by the aquaporin-like pentameric formate channel FocA of E. coli is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate (Pinske and Sawers 2016).

The phylogenetic tree shows clustering according to function and organismal phylogeny. The putative formate efflux transporters (FocA) of bacteria associated with pyruvate-formate lyase (pfl) comprise cluster I; the putative formate uptake permeases (FdhC) of bacteria and archaea associated with formate dehydrogenase comprise cluster II; the nitrite uptake permeases (NirC) of bacteria comprise cluster III, and a yeast protein comprises cluster IV (see Mukherjee et al. 2017.

The energy coupling mechanisms for proteins of the FNT family have not been extensively characterized. HCO2- and NO2- uptakes are probably coupled to H+ symport. HCO2- efflux may be driven by the membrane potential by a uniport mechanism or by H+ antiport. FocA of E. coli catalyzes bidirectional formate transport and may function by a channel-type mechanism (Falke et al., 2010).

FocA homologues transports short-chain acids in bacteria, archaea, fungi, algae and certain eukaryotic parasites. Wang et al. (2009) reported the crystal structure of the E. coli FocA at 2.25 Å resolution. FocA forms a symmetric pentamer, with each protomer consisting of six TMSs. Despite a lack of sequence homology, the overall structure of the FocA protomer closely resembles that of aquaporin, indicating that FocA is a channel rather than a carrier. Structural analysis identified potentially important channel residues, defined the channel path and revealed two constriction sites. Unlike aquaporin, FocA is impermeable to water but allows the passage of formate.

FocA (2.A.44.1.1) may be able to switch its mode of operation from a passive export channel at high external pH to a secondary active formate/H+ importer at low pH. The crystal structure of Salmonella typhimurium FocA at pH 4.0 shows that this switch involves a major rearrangement of the amino termini of individual protomers in the pentameric channel (et al., 2011).The amino-terminal helices open or block transport in a concerted, cooperative action that indicates how FocA is gated in a pH-dependent way. Electrophysiological studies show that the protein acts as a specific formate channel at pH 7.0 and that it closes upon a shift of pH to 5.1.

Phylogenetic analysis of prokaryotic FNT sequences revealed eight different subgroups (Mukherjee et al. 2017). Formate, nitrite and hydrosulphide transporters respectively are clustered into two (FocA and FdhC), three (NirC-alpha, NirC-beta and NirC-gamma) and one (HSC) subfamilies plus two FNT subgroups (YfdC-alpha and YfdC-beta) with unassigned function. Structure-based sequence alignments of individual subfamily members revealed that certain positions in the two constriction regions and some residues facing the interior show subfamily-specific conservation (Mukherjee et al. 2017).

Transmembrane transport of monocarboxylates is conferred by structurally diverse membrane proteins. Bader and Beitz 2020 described the pH dependence of lactic acid/lactate facilitation of an aquaporin (AQP9, TC# 1.A.8.9.14), a monocarboxylate transporter (MCT1, SLC16A1, TC# 2.A.1.13.1), and a formate-nitrite transporter (Plasmodium falciparum FNT, PfFNT, TC# 1.A.16.2.7) in the equilibrium transport state. FNTs exhibit a channel-like structure mimicking the aquaporin-fold, yet act as secondary active transporters. Bader and Beitz 2020 used radiolabeled lactate to monitor uptake via yeast-expressed AQP9, MCT1, and PfFNT for long enough time periods to reach the equilibrium state in which import and export rates are balanced. They confirmed that AQP9 behaved perfectly equilibrative for lactic acid, i.e., the neutral lactic acid molecule enters and passes the channel. MCT1, in turn, actively used the transmembrane proton gradient and acted as a lactate/H+ co-transporter. PfFNT behaved highly similar to the MCT in terms of transport properties, although it did not adhere to the classical alternating access transporter model. Instead, FNT appears to use the proton gradient to neutralize the lactate anion in the protein's vestibule to generate lactic acid in a place that traverses the central hydrophobic transport path. Thus, they proposed to include FNT-type proteins into a more generalized, function-based transporter definition (Bader and Beitz 2020).

The probable transport reactions catalyzed by different members of the FNT family are:

(1) RCO2- or NO2- (out) ⇌ RCO2- or NO2- (in)

(2) HCO2- (in) ⇌ HCO2- (out)

(3) HS- (out) ⇌ HS- (in)

This family belongs to the: Major Intrinsic Protein (MIP) Superfamily.

References associated with 1.A.16 family:

Andrews, S.C., B.C. Berks, J. McClay, A. Ambler, M.A. Quail, P. Golby, and J.R. Guest. (1997). A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology 143(Pt11): 3633-3647. 9387241
Atkovska, K. and J.S. Hub. (2017). Energetics and mechanism of anion permeation across formate-nitrite transporters. Sci Rep 7: 12027. 28931899
Bader, A. and E. Beitz. (2020). Transmembrane Facilitation of Lactate/H Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability. Membranes (Basel) 10:. 32942665
Czyzewski, B.K. and D.N. Wang. (2012). Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483: 494-497. 22407320
Davies, H., B. Bergmann, P. Walloch, C. Nerlich, C. Hansen, S. Wittlin, T. Spielmann, M. Treeck, and E. Beitz. (2023). The Lactate/H Transporter PfFNT Is Essential and Druggable. Antimicrob. Agents Chemother. e0035623. [Epub: Ahead of Print] 37428074
Doberenz, C., M. Zorn, D. Falke, D. Nannemann, D. Hunger, L. Beyer, C.H. Ihling, J. Meiler, A. Sinz, and R.G. Sawers. (2014). Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation. J. Mol. Biol. 426: 2827-2839. 24887098
Falke, D., C. Doberenz, D. Hunger, and R.G. Sawers. (2016). The glycyl-radical enzyme 2-ketobutyrate formate-lyase, TdcE, interacts specifically with the formate-translocating FNT-channel protein FocA. Biochem Biophys Rep 6: 185-189. 28955877
Falke, D., K. Schulz, C. Doberenz, L. Beyer, H. Lilie, B. Thiemer, and R.G. Sawers. (2010). Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate-nitrite transporter family of integral membrane proteins. FEMS Microbiol. Lett. 303: 69-75. 20041954
Golldack, A., B. Henke, B. Bergmann, M. Wiechert, H. Erler, A. Blancke Soares, T. Spielmann, and E. Beitz. (2017). Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale. PLoS Pathog 13: e1006172. 28178358
Hapuarachchi, S.V., S.A. Cobbold, S.H. Shafik, A.S. Dennis, M.J. McConville, R.E. Martin, K. Kirk, and A.M. Lehane. (2017). The Malaria Parasite''s Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens. PLoS Pathog 13: e1006180. 28178359
Helmstetter, F., P. Arnold, B. Höger, L.M. Petersen, and E. Beitz. (2019). Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport. J. Biol. Chem. 294: 623-631. 30455351
Hunger, D., M. Röcker, D. Falke, H. Lilie, and R.G. Sawers. (2017). The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity. Front Microbiol 8: 1616. 28878762
Jia, W., N. Tovell, S. Clegg, M. Trimmer, and J. Cole. (2009). A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake. Biochem. J. 417: 297-304. 18691156
Kammel, M., O. Trebbin, and R.G. Sawers. (2022). Interplay between the Conserved Pore Residues Thr-91 and His-209 Controls Formate Translocation through the FocA Channel. Microb Physiol. [Epub: Ahead of Print] 35390794
Kuzminov, A. and F.W. Stahl. (1997). Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation. J. Bacteriol. 179: 880-888. 9006046
Lü, W., J. Du, T. Wacker, E. Gerbig-Smentek, S.L. Andrade, and O. Einsle. (2011). pH-dependent gating in a FocA formate channel. Science 332: 352-354. 21493860
Lyu, M., C.C. Su, J.W. Kazura, and E.W. Yu. (2021). Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT. EMBO Rep e51628. [Epub: Ahead of Print] 33471955
Moraes, T.F. and R.A. Reithmeier. (2012). Membrane transport metabolons. Biochim. Biophys. Acta. 1818: 2687-2706. 22705263
Mukherjee, M., M. Vajpai, and R. Sankararamakrishnan. (2017). Anion-selective Formate/nitrite transporters: taxonomic distribution, phylogenetic analysis and subfamily-specific conservation pattern in prokaryotes. BMC Genomics 18: 560. 28738779
Nakata, K., M.M. Koh, T. Tsuchido, and Y. Matsumura. (2010). All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant. Appl. Microbiol. Biotechnol. 87: 1895-1905. 20480162
Nölling, J. and J.N. Reeve. (1997). Growth- and substrate-dependent transcription of the formate dehydrogenase (fdhCAB) operon in Methanobacterium thermoformicicum Z-245. J. Bacteriol. 179: 899-908. 9006048
Park, J.S., S.J. Lee, H.G. Rhie, and H.S. Lee. (2008). Characterization of a chromosomal nickel resistance determinant from Klebsiella oxytoca CCUG 15788. J Microbiol Biotechnol 18: 1040-1043. 18600044
Pinske, C. and R.G. Sawers. (2016). Anaerobic Formate and Hydrogen Metabolism. EcoSal Plus 7:. 27735784
Pui, C.H., W.M. Crist, and A.T. Look. (1990). Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood 76: 1449-1463. 2207320
Rycovska-Blume, A., W. Lü, S. Andrade, K. Fendler, and O. Einsle. (2015). Structural and Functional Studies of NirC from Salmonella typhimurium. Methods Enzymol 556: 475-497. 25857796
Schmidt, J.D.R. and E. Beitz. (2021). Mutational Widening of Constrictions in a Formate-Nitrite/H Transporter Enables Aquaporin-Like Water Permeability and Proton Conductance. J. Biol. Chem. 101513. [Epub: Ahead of Print] 34929166
Unkles, S.E., K.L. Hawker, C. Grieve, E.I. Campbell, P. Montague, and J.R. Kinghorn. (1991). crnA encodes a nitrate transporter in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 88: 204-208. 1986367
Unkles, S.E., V.F. Symington, Z. Kotur, Y. Wang, M.Y. Siddiqi, J.R. Kinghorn, and A.D. Glass. (2011). Physiological and biochemical characterization of AnNitA, the Aspergillus nidulans high-affinity nitrite transporter. Eukaryot. Cell. 10: 1724-1732. 22021238
Waight, A.B., J. Love, and D.N. Wang. (2010). Structure and mechanism of a pentameric formate channel. Nat Struct Mol Biol 17: 31-37. 20010838
Wang, Y., Y. Huang, J. Wang, C. Cheng, W. Huang, P. Lu, Y.N. Xu, P. Wang, N. Yan, and Y. Shi. (2009). Structure of the formate transporter FocA reveals a pentameric aquaporin-like channel. Nature 462: 467-472. 19940917
Wiechert, M., H. Erler, A. Golldack, and E. Beitz. (2017). A widened substrate selectivity filter of eukaryotic formate-nitrite transporters enables high-level lactate conductance. FEBS J. 284: 2663-2673. 28544379
Wood, G.E., A.K. Haydock, and J.A. Leigh. (2003). Function and regulation of the formate dehydrogenase genes of the methanogenic archaeon Methanococcus maripaludis. J. Bacteriol. 185: 2548-2554. 12670979