The allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

This work reports the evolutionary relationships, amplified expression, functional characterisation and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI 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 PucI were consistent with twelve putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions are described for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5% in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energised Escherichia coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3M, therefore confirming that PucI is a medium affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognises some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilised from inner membranes using n-dodecyl-β-D-maltoside and purified. The isolated protein comprised a substantial proportion of α-helix secondary structure, consistent with the predictions, and a three-dimensional model was therefore constructed on a template of the Mhp1 structure, which aided localisation of the potential ligand binding site in PucI.