1887

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are relatively common prokaryotic secondary transporters which comprise an extracytoplasmic solute receptor (ESR) protein and two dissimilar membrane proteins or domains, yet the substrates and physiological functions of only a few of these systems are so far known. In this study, a biophysical approach was used to identify the ligands for the purified RRC01191 and YiaO proteins, which are members of two phylogenetically distinct families of TRAP-ESRs found in diverse bacteria. In contrast to previous indirect evidence pointing to RRC01191 orthologues being involved in polyol uptake, it was shown that RRC01191 binds pyruvate, 2-oxobutyrate and a broad range of aliphatic monocarboxylic 2-oxoacid anions with varying affinities ( values 0·08–3 μM), consistent with a predicted role in monocarboxylate transport related to branched-chain amino-acid biosynthesis. The YiaMNO TRAP transporter has previously been proposed to be an -xylulose uptake system [ Plantinga . (2004) , 51–57], but purified YiaO did not bind - or -xylulose as judged by fluorescence spectroscopy, circular dichroism or mass spectrometry. Instead, these techniques showed that a breakdown product of -ascorbate, 2,3-diketo--gulonate (2,3-DKG), binds by a simple one-step mechanism with sub-micromolar affinity. The data provide the first evidence for the existence of ESR-dependent transporters for 2-oxoacids and 2,3-DKG, homologues of which appear to be widespread amongst prokaryotes. The results also underline the utility of direct ESR ligand-binding studies for TRAP transporter characterization

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2006-01-01
2024-12-14
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