Biol. Chem., Vol. 389, pp. 33–36, January 2008 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.002 2008/254 Article in press - uncorrected proof Short Communication A new subfamily of bacterial glutamate/aspartate receptors Birendra Singh* and Klaus-Heinrich Ro ¨ hm Institute of Physiological Chemistry, Philipps University, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany * Corresponding author e-mail: singh@staff.uni-marburg.de Abstract The specificity of bacterial nutrient importers of the ATP- binding cassette (ABC) type depends on external recep- tor proteins that not only bind the solute to be transported, but also initiate the transport process by inducing ATP hydrolysis in the cytoplasmic nucleotide- binding domains. Here we propose a mode of ligand binding to the solute-binding protein AatJ that is required for glutamate uptake by the AatJMQP transporter in Pseudomonas putida KT2440. A homology model of the AatJ-glutamate complex was constructed using the E. coli glutamine-binding protein GlnH as the template. The general validity of the model was then confirmed by alanine scanning mutagenesis of several residues pre- dicted to interact with the ligand and by semi-quantitative binding studies with w 14 Cx -Glu and w 14 Cx -Asp. A database search indicated that AatJ is a member of a distinct sub- family of the family 3 solute-binding proteins with spec- ificity towards glutamate and aspartate. Keywords: AatJ; ABC transporter; glutamate; Pseudomonas putida; solute-binding protein. ATP-binding cassette (ABC) transporters are a large superfamily of proteins that occur in all organisms and translocate solutes across biological membranes in an ATP-dependent fashion (Higgins, 1992). Most ABC transporters are made up of four components: two membrane-spanning domains and two intracellular nucleotide-binding domains (NBDs). In eukaryotes, these modules are usually organized as a single polypeptide chain, whereas bacterial ABC transporters are assem- bled from individual subunits. Bacterial systems that import extracellular solutes require an additional solute- specific binding protein (or solute receptor). These recep- tors reside outside the cell membrane (in the periplasmic space of Gram-negative bacteria or associated with the cell surface of Gram-positive organisms) and are crucial components of the transport machinery. They not only define the specificity of transport, but also stimulate the ATPase activity of the NBDs once they have bound their cognate solute, thereby initiating the translocation pro- cess (Davidson and Chen, 2004). Recently, we identified in Pseudomonas putida KT2440 an ABC transporter (termed Aat for acidic amino acid transport) that is indispensable for the utilization of glu- tamate and glutamine by this strain. Both amino acids are excellent substrates for P. putida, allowing growth rates that are comparable to or higher than those reached on NH 4 q and glucose (Sonawane et al., 2003, 2006). As usual, the Aat transporter, which is encoded by genes PP1068–PP1071, involves two different per- mease subunits (AatM and AatQ), an NBD (AatP), and an extracellular solute receptor, AatJ. Closely similar sys- tems also exist in P. aeruginosa (PA1339–PA1342), P. fluorescens (Pfl1048–Pfl1051) and P. syringae (PSPTO4171–PSPTO4174). Mutants with inactivated AatJ were unable to grow on Glu or Gln, whereas growth on Asp or Asn was only moderately impaired (Singh et al., 2007). As P. putida KT2440 does not contain an uptake system for Gln, this amino acid has first to be converted to glutamate by a periplasmic glutaminase/ asparaginase (Hu ¨ ser et al., 1999). Thus, the observation that AatJ is required for the utilization of both Glu and Gln indicates that the AatJMQP transporter is the only high-affinity glutamate importer of strain KT2440. Based on sequence similarities, Tam and Saier (1993) have subdivided the group of bacterial extracellular sol- ute-binding proteins (SBP) into eight families or clusters. According to this system, AatJ belongs to SBP cluster 3, which groups together amino acid- and opine-binding receptors. In the present communication we demonstrate that AatJ and at least 10 further bacterial solute receptors form a subfamily within SBP cluster 3 that includes receptors with a marked selectivity for acidic amino acids (Glu and Asp). This suggestion, which originated from a homology model of the AatJ amino acid-binding site, is supported by the amino acid-binding properties of vari- ous AatJ mutants and by an analysis of the known SBP family 3 sequences. To date, crystal structures of three SBP cluster 3 pro- teins are available: GlnH from E. coli (PDB entries 1ggg and 1wdn), HisJ from Salmonella typhimurium (1hpp), and the arginine/lysine-binding protein from the same organism (1laf, 1lag and 2lao). The modes of ligand bind- ing to these proteins were compared in detail by Sun et al. (1998). In all known SBP family 3 structures, the car- boxylate group of the bound amino acid is stabilized by electrostatic interactions with an arginine residue that is strictly conserved in all family 3 proteins (in AatJ num- bering, used here and in the following, this is R93). In addition, a threonine or serine residue at position -5 rel- ative to this arginine interacts with the a-carboxylate and/ or a-ammonium group of the bound ligand (T88 in AatJ). As expected, the environment of the side chains of the bound ligands (Gln, His, and Lys, respectively) are as dif- ferent as the side chains themselves (Sun et al., 1998). 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