A Bacterial Arginine-Agmatine Exchange Transporter Involved in Extreme Acid Resistance

Abstract

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of ϳ10 mg/liter bacterial culture. Glutaraldehyde crosslinking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K m ϳ80 M for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

Molecular basis of substrate-induced permeation by an amino acid antiporter

“…This result was surprising because both mutants exchange L-arginine ( Table 1). The same experiments performed with wild-type AdiC and N101D gave, in both cases, similar apparent Kd values (∼100 μM) (Table 1), as previously described for wild type (3,4). Because N101A and W293Y recognize L-arginine but have serious defects in translocation, particularly the former, it is possible that the heat measured during AdiC-L-arginine binding is associated mostly with the conformational changes induced by the substrate.…”

“…T he antiporter AdiC is a virtual proton pump that expels protons in Escherichia coli and other enteric bacteria in extremely acid environments (1,2) by exchanging extracellular L-arginine (Arg þ ) for intracellular agmatine (Agm 2þ ) (3,4). AdiC is a member of the basic Amino acid/Polyamine Antiporter (APA) family within the amino acid, Polyamine, and organic Cation (APC) superfamily of transporters (5).…”

“…At present, the crystal structures of AdiC (17)(18)(19) and the broad-specificity amino acid transporter ApcT (20) are the closest structural paradigms for CATs and LATs (<20% amino acid identity). AdiC is a homodimer (3,4) in which each protomer contains the "5 þ 5 inverted repeat" fold (17,18), initially described in Na þ -coupled symporters (21)(22)(23)(24). Mechanistically, the AdiC structures of the wild-type and the double mutant N22A-L123W represent two conformational states within the transport cycle: the open-toout apo (PDB 3LRB and 3NCY) (17,18) and the outward-facing Arg þ -bound occluded (PDB 3L1L) (19), respectively.…”

Molecular mechanism of pH-dependent substrate transport by an arginine-agmatine antiporter

“…In E. coli, AR2 and AR3 each use two molecular components, a membrane-embedded amino acid antiporter and a cytosolic decarboxylase, to consume and expel intracellular protons (1)(2)(3). AR3 consists of the antiporter AdiC, which exchanges extracellular L-arginine (Arg) with intracellular agmatine (Agm), and the decarboxylase AdiA, which converts Arg to Agm by removing a carbon dioxide molecule from the α-carboxylate group of Arg and thus absorbing an intracellular proton (2)(3)(4)(5)(6). Similarly, AR2 comprises an L-glutamate (Glu):γ-aminobutyric acid (GABA) antiporter GadC and two Glu decarboxylases, GadA and GadB, which convert Glu to GABA (7,8).…”

“…The transport activity of the amino acid antiporter AdiC or GadC is strictly pH-dependent (6,9). Both transporters display robust transport activity only at pH values of 6.0 or lower.…”