Allantoin transport protein, Pucl, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, Pucl, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between Pucl and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for Pucl were consistent with 12 putative transmembrane-spanning alpha-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucl gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the Pucl(His(6)) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible Pucl-mediated uptake of C-14-allantoin into energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (K(m)app) of 24 +/- 3 mu M, therefore confirming that Pucl is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that Pucl recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. Pucl(His(6)) was solubilized from inner membranes using n-dodecyl-beta-D-maltoside and purified. The isolated protein contained a substantial proportion of alpha-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in Pucl.