2.A.81 The Aspartate:Alanine Exchanger (AAEx) Family
A single functionally characterized protein, the aspartate:alanine exchanger (AspT) of the Gram-positive lactic acid bacterium, Tetragenococcus halophila D10 serves to characterize the AAEx family (Abe et al., 2002). This organism takes up L-aspartate, decarboxylates it to L-alanine and CO2 in the cytoplasm, catalyzed by L-aspartate β-decarboxylase (AspD), and exports the L-alanine in a 1:1 exchange reaction with L-aspartate. AspT is a hydrophobic protein of 543 aas and 10 putative TMSs with two TrkA-C domains between TMSs 5 and 6. This protein has many Gram-negative and Gram-positive bacterial homologues of unknown function, and possibly one very distant homologue in the archaeon, Halobacterium sp. strain NRC-1. This protein (384 aas; 10-12 putative TMSs; AAC82885) includes a 54-residue region (residues 6-60) that shows 35% identity and 51% similarity with the ammonium transporter, Amt of Corynebacterium glutamicum (spP54146). These proteins exhibit an internal duplication with 5 or 6 TMSs per repeat element.
Because one more negative charge is brought in (aspartate) that is exported (alanine), the exchange transport process results in net charge movement, creating a membrane potential, negative inside. Further, decarboxylation of aspartate consumes a scalar proton and thus generates a pH gradient (basic inside). The resultant pmf can drive ATP synthesis via the F-type ATPase (TC #3.A.2). Other such exchangers generating a pmf are the prototypical oxalate/formate exchanger of the MFS (TC #3.A.1) as well as glutamate/γ-amino butyrate, malate/lactate, citrate/lactate and histidine/histamine exchangers (for references see Abe et al., 2002).
AspT has 10 transmembrane helices (TMS), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3 (Nanatani et al., 2007). The hydrophilic cytoplasmic loop of AspT possesses a sequence divergent TrkA_C domain.
The generalized transport reaction catalyzed by AspT is:
L-aspartate (out) + L-alanine (in) ⇌ L aspartate (in) + L-alanine (out)
References:
The L-aspartate:L-alanine exchanger, AspT (Abe et al. 1996; Abe et al. 2002). A mutant, R76K, has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than AspT-WT. Thus, R76 is involved in AspT substrate transport (Suzuki et al. 2016). AspT catalyzes self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high (Abe et al. 2002). AspT has been reported to have a unique topology; 8 TMS, a large cytoplasmic loop (183 amino acids) between TMS5 and TMS6, and N- and C-termini that both face the periplasm. This may be a unique 2D-structure of AspT, a representative of the novel AAE family (Nanatani et al. 2005). However, bioinformatic analyses suggest that this prediction may be in error, and the true topology is 12 TMSs with an internal repeat of 6 TMSs (M. Saier, unpublished observations; see 2.A.81.2.1). The Km values = 0.35 mm for L-aspartate, 0.098 mm for D-aspartate, 26 mm for L-alanine, and 3.3 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange while L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT (Sasahara et al. 2011). L-Ala binding yields a conformation different from the apo or L-Asp binding conformations (Suzuki et al. 2022).
Bacteria and archaea
AspT of Pediococcus halophila (Tetragenococcus halophila) sp. D10 (Q8L3K8)
The putative cobalt porter, CbtD (Rodionov et al. 2003)
Bacteroidetes
CbtD of Bacteroides fragilis (Q5LCC7)
Succinate/ibuprofin/taurine transporter, SucE1 or TMEM184B. Its expression is induced under microaerobic and anaerobic conditions when succinate is produced from glucose via the reductive tricarboxylic acid cycle, and it exhibits succinate counterflow (self exchange) (Fukui et al., 2011).
Bacteria
SucE1 of Corynebacterium glutamicum (Q8NNI8)
Putative transporter, YbjL
Bacteria
YbjL of E. coli
Bacteria
YidE of Escherichia coli
Aspartate:alanine antiporter, AspT, of 561 aas. AspT has 7 TMSs, a large cytoplasmic loop containing approximately 200 aas between TMS4 and TMS5, a cytoplasmic N-terminus, and a periplasmic C-terminus (Fujiki et al. 2007).
AspT of Pseudomonas dacunhae (Comamonas testosteroni)
Homologue of AspT (384 aas; 12 putative TMSs; with potential membrane embedded loops between TMSs 5 and 6 and TMSs 11 and 12 (the two halves are internally duplicated). The central hydrophilic domain in 2.A.81.1.1 is absent in this homologue. Although the experimentally predicted topology for 2.A.81.1.1 differs from this prediction, we consider it possible that the experimentally determined topology is in error (M. Saier, unpublished observations).
Archaea
AspT homologue of Halobacterium sp. NRC-1 (AAC82885)
YidE/YbjL duplication protein
Thermotogae
YidE homologue of Thermosipho melanesiensis