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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)

References associated with 2.A.24 family:

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. 10441129
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. 10993891
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. 9218448
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. 9457870
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. 19594131
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. 12029396
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. 9190827
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. 23810698
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. 11566984
Pudlik, A.M. and J.S. Lolkema. (2012). Substrate specificity of the citrate transporter CitP of Lactococcus lactis. J. Bacteriol. 194: 3627-3635. 22563050
Reizer, J., A. Reizer, and M.H. Saier, Jr. (1994). A functional superfamily of sodium/solute symporters. Biochim. Biophys. Acta 1197: 133-166. 8031825
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. 11948157
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. 15148311
Sobczak, I. and J.S. Lolkema. (2005). Loop VIII/IX of the Na+-citrate transporter CitS of Klebsiella pneumoniae folds into an amphipathic surface helix. Biochemistry 44: 5461-5470. 15807539
Sobczak, I. and Lolkema, J.S. (2005). The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism. Microbiol. Mol. Biol. Rev. 69: 665-695. 16339740
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. 10514443
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. 10825453