2.A.24 The 2-Hydroxycarboxylate Transporter (2-HCT) Family
Members of 2-HCT family catalyze citrate or malate uptake with either Na+ or H+ as the cotransported cation or substrate:decarboxylated product antiport (Sobczak and Lolkema, 2005). However, three functionally characterized members, MaeP of Streptococcus bovis, CitP of Leuconostoc mesenteroides and CitW of Klebsiella pneumoniae are malate:lactate, citrate:lactate and citrate:acetate antiporters, respectively. A single arginyl residue, Arg-425 on the inner side of TMSII in CitP, binds one of the carboxylates in a dicarboxylate substrate but does not bind the carboxylate in a monocarboxylate substrate (Bandell and Lolkema, 2000). This shows that the C-terminal domain is involved in substrate binding. CitW of K. pneumoniae transports [H+ · citrate]-2 in exchange for the product of citrate fermentation, acetate, and is expressed only under anoxic conditions (Kästner et al., 2002).
The proteins of the 2-HCT family have been found in both Gram-negative and Gram-positive bacteria but not in other organisms. However, regions of weak sequence similarity are observed in an archaeal protein (gi 7518744) which proves to be a member of the DMT superfamily (most like the protein of TC #2.A.7.3.5). 2-HCT family members contain about 450 amino acyl residues and possess 10-12 putative transmembrane helical spanners. An eleven TMS topology has been experimentally documented for the Klebsiella pneumoniae CitS protein. These proteins contain two repeat units of 5 TMSs with membrane-inserted loop (pore-loop structure) between TMSs 10 and 11, and possibly one between TMSs 5 and 6. These regions, which enter the membrane from opposite sides of the membrane, may line the channel and influence substrate binding (Sobczak and Lolkema, 2004).
Dobrowolski and Lolkema (2009) have pointed to structural and mechanistic similarities between the ESS (TC #2.A.27) and 2-HCT (TC #2.A.24)
transporters, as well as the two domain structure of the
transporters and the presence and functional importance of the
reentrant loops present in both domains. They propose that the conserved GGXG motifs
are at the vertex of the reentrant loops.
The generalized transport reactions for the members of this family are:
Di- or tricarboxylate (out) + n[H+ or Na+] (out) → Di- or tricarboxylate (in) + n[H+ or Na+] (in)
Di- or Tricarboxylate (out) + Mono- or dicarboxylate (in) → Di- or Tricarboxylate (in) + Mono- or dicarboxylate (out)
This family belongs to the .
|Bandell, M. and J.S. Lolkema. (1999). Stereoselectivity of the membrane potential-generating citrate and malate transporters of lactic acid bacteria. Biochemistry 38: 10352-10360.|
|Bandell, M. and J.S. Lolkema. (2000). Arg-425 of the citrate transporter CitP is responsible for high affinity binding of di- and tricarboxylates. J. Biol. Chem. 275: 39130-39136.|
|Bandell, M., V. Ansanay, N. Rachidi, S. Dequin, and J.S. Lolkema. (1997). Membrane potential-generating malate (MleP) and citrate (CitP) transporters of lactic acid bacteria are homologous proteins - Substrate specificity of the 2-hydroxycarboxylate transporter family. J. Biol. Chem. 272: 18140-18146.|
|Bekal, S., J. Van Beeuman, B. Samyn, D. Garmyn, S. Henini, C. Divies, and H. Prévost. (1998). Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster. J. Bacteriol. 180: 647-654.|
|Dobrowolski A. and Lolkema JS. (2009). Functional importance of GGXG sequence motifs in putative reentrant loops of 2HCT and ESS transport proteins. Biochemistry. 48(31):7448-56.|
|Kästner, C.N., K. Schneider, P. Dimroth, K.M. Pos. (2002). Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae. Arch. Microbiol. 177: 500-506.|
|Kawai, S., H. Suzuki, K. Yamamoto, and H. Kumagai. (1997). Characterization of the L-malate permease gene (maeP) of Streptococcus bovisATCC 15352. J. Bacteriol. 179: 4056-4060.|
|Kebbel F., Kurz M., Arheit M., Grutter MG. and Stahlberg H. (2013). Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography. Structure. 21(7):1243-50.|
|Krom, B.P., R. Aardema, and J.S. Lolkema. (2001). Bacillus subtilisYxkJ is a secondary transporter of the 2-hydroxycarboxylate transporter family that transports L-malate and citrate. J. Bacteriol. 183: 5862-5869.|
|Pudlik, A.M. and J.S. Lolkema. (2012). Substrate specificity of the citrate transporter CitP of Lactococcus lactis. J. Bacteriol. 194: 3627-3635.|
|Reizer, J., A. Reizer, and M.H. Saier, Jr. (1994). A functional superfamily of sodium/solute symporters. Biochim. Biophys. Acta 1197: 133-166.|
|Schneider, K., C.N. Kästner, M. Meyer, M. Wessel, P. Dimroth, and M. Bott. (2002). Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group. J. Bacteriol. 184: 2439-2446.|
|Sobczak, I. and J.S. Lolkema. (2004). Alternating access and a pore-loop structure in the Na+-citrate transporter CitS of Klebsiella pneumoniae. J. Biol. Chem. 279: 31113-31120. |
|Sobczak, I. and Lolkema, J.S. (2005). The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism. Microbiol. Mol. Biol. Rev. 69: 665-695. |
|van Geest, M. and J.S. Lolkema. (1999). Transmembrane segment (TMS) VIII of the Na+/citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia colimembrane. J. Biol. Chem. 274: 29705-29711.|
|van Geest, M. and J.S. Lolkema. (2000). Membrane topology of the Na(+)/citrate transporter CitS of Klebsiella pneumoniae by insertion mutagenesis. Biochim. Biophys. Acta 1466: 328-338.|
Citrate:Na+ symporter, CitS. Kebbel et al. 2013 presented the three-dimensional map of dimeric CitS
obtained with electron crystallography. Each monomer has 13 alpha-helical transmembrane segments;
six are organized in a distal helix cluster and seven in the central dimer interface domain. Based
on structural analyses and comparison to VcINDY, a molecular model with assigned
helices, a model with internal structural symmetry was proposed. Projections of CitS in
several conformational states induced by the presence and absence of sodium and citrate as
substrates were also proposed. Citrate binding induces a defined movement of alpha helices within the distal helical
cluster. Kebbel et al. 2013 proposed a substrate translocation site and conformational changes that
are in agreement with the "alternating access" model.
CitS of Klebsiella pneumoniae
|2.A.24.2.1||L-Malate permease ||Bacteria ||MaeP of Streptococcus bovis |
|2.A.24.2.2||Malate:lactate antiporter (substrates include: S-lactate, R-lactate, S-malate and S-citramalate) ||Bacteria ||MaeP of Lactococcus lactis |
|2.A.24.2.3||Malate:Na+ symporter ||Bacteria ||YufR of Bacillus subtilis |
|2.A.24.2.4||L-malate/citrate:H+ symporter (electroneutral)||Bacteria||CimH (YxkJ) of Bacillus subtilis |
|2.A.24.2.5||Citrate:acetate antiporter, CitW||Bacteria||CitW of Klebsiella pneumoniae|
Electrogenic citrate:L-lactate exchanger, CitP or CitN (Pudlik and Lolkema 2012).
CitN of Lactococcus lactis
|2.A.24.3.2||Citrate:lactate antiporter (substrates include: citrate, S-citramalate, S-malate, 2-hydroxylisobutyrate and R-lactate) ||Bacteria ||CitP of Leuconostoc mesenteroides|