Determinants of substrate specificity and biochemical properties of the sn‐glycerol‐3‐phosphate ATP binding cassette transporter (UgpB–AEC₂) of Escherichia coli

Abstract

SummaryUnder phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 Ϯ 0.02 mM and 5.1 Ϯ 0.3 mM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC 2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable.

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

“…This can be ascribed to some extent to the fact that GlpT is a G3P importer at the price of simultaneously being a phosphate exporter (63). In agreement with this notion, previous studies suggested that Glp-transported G3P can serve as the sole source for both carbon and phosphate, whereas Ugp-transported G3P can serve as the sole source only for phosphate (62,64). Thus, we believe that Ugp induction is mostly driven by phosphate starvation, whereas the Glp repression is likely due to unavailability of G3P as a carbon source as well as to phosphate limitation.…”

“…As one of two major sn-glycerol-3-phosphate (G3P) uptake systems, the ugp operon is often induced when bacteria are starved for inorganic phosphate (62). Interestingly, while the Ugp system was induced in response to phosphate limitation, the other G3P transporter, the Glp system, was significantly repressed at 6 h postinfection.…”

Ugp and PitA Participate in the Selection of PHO-Constitutive Mutants

“…Another possibility would be that G2P is converted into G3P in the periplasm and only then is transported by Ugp. This may be in agreement with a recent report that challenged the notion that Ugp is able to take up G2P in vitro (23). However, a periplasmic enzyme that catalyzes the conversion of G2P to G3P is still unknown.…”

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

“…PWBP57 has a two-domain structure containing an N-terminal UgpB domain and a C-terminal conserved domain of unknown function (DUF3502). The UgpB protein, a member of the SBP superfamily, binds to sn-glycerol-3-phosphate and is structurally similar to SBPs (29). Sequence identity (in amino acid residues) was 13 to 32% among the SBP domains of PWBPs.…”

“…The UgpB domain belongs to cluster B in the SBP superfamily and is structurally similar to SBPs (29). Most SBPs with specific binding to saccharides are classified in cluster B and are a component of ATP-binding cassette (ABC) transporters for substrate uptake.…”