Thomas Gavin H, Southworth Thomas, León-Kempis Maria Rocio, Leech Andrew, Kelly David J
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Microbiology (Reading). 2006 Jan;152(Pt 1):187-198. doi: 10.1099/mic.0.28334-0.
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 Rhodobacter capsulatus RRC01191 and Escherichia coli 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 (K(d) values 0.08-3 muM), consistent with a predicted role in monocarboxylate transport related to branched-chain amino-acid biosynthesis. The E. coli YiaMNO TRAP transporter has previously been proposed to be an l-xylulose uptake system [Plantinga et al. (2004) Mol Membr Biol 21, 51-57], but purified YiaO did not bind l- or d-xylulose as judged by fluorescence spectroscopy, circular dichroism or mass spectrometry. Instead, these techniques showed that a breakdown product of l-ascorbate, 2,3-diketo-l-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.
不依赖ATP的周质三方（TRAP）转运蛋白是相对常见的原核生物二级转运蛋白，由胞外溶质受体（ESR）蛋白以及两个不同的膜蛋白或结构域组成，然而迄今为止，这些系统中只有少数几个的底物和生理功能是已知的。在本研究中，采用生物物理方法来鉴定纯化的荚膜红细菌RRC01191蛋白和大肠杆菌YiaO蛋白的配体，它们是在不同细菌中发现的两个系统发育上不同的TRAP-ESR家族的成员。与之前指向RRC01191直系同源物参与多元醇摄取的间接证据相反，研究表明RRC01191以不同亲和力（解离常数K(d)值为0.08 - 3 μM）结合丙酮酸、2-氧代丁酸以及一系列脂肪族单羧酸2-氧代酸阴离子，这与在支链氨基酸生物合成相关的单羧酸转运中的预测作用一致。大肠杆菌YiaMNO TRAP转运蛋白先前被认为是一种L-木酮糖摄取系统[Plantinga等人（2004年），《分子膜生物学》21卷，51 - 57页]，但通过荧光光谱、圆二色性或质谱判断，纯化的YiaO并不结合L-或D-木酮糖。相反，这些技术表明L-抗坏血酸的一种分解产物2,3-二酮-L-古洛糖酸（2,3-DKG）通过简单的一步机制以亚微摩尔亲和力结合。这些数据首次证明了存在依赖ESR的2-氧代酸和2,3-DKG转运蛋白，其同源物似乎在原核生物中广泛存在。结果还强调了直接的ESR配体结合研究在TRAP转运蛋白表征中的实用性。
