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 (Lü 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).
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)