Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family

This paper describes the functional characterization of the xyloside transp orter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xylo side isoprimeverose, a building block of hemicellulose, and the analogue me thyl-isoprimeverose were chemically synthesized by a new and efficient proc edure. The XylP protein was purified by metal affinity chromatography, foll owing high level expression in Lactococcus lactis from. the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in wh ich the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was e ntrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol- indophenol as electron acceptor. The coupled assay thus involves XyIP-media ted isoprimeverose uptake followed by internal oxidation of the sugar by sG DH, which can be monitored from the reduction of 2,6-dichlorophenol-indophe nol at 600 run. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membr ane, indicating that exchange transport is faster than unidirectional downh ill uptake. Unlike other members of the galactoside-pentoside-hexuronide fa mily, XylP does not transport monosaccharides (xylose) but requires a glyco sidic linkage at the anomeric carbon position. Consistent with a proton mot ive force-driven mechanism, the uptake was stimulated by a membrane potenti al (inside negative relative to outside) and inhibited by a pH gradient (in side acidic relative to outside). The advantages of the here-described tran sport assay for studies of carbohydrate transport are discussed.