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

Pikyee Ma,Peter J F Henderson,Simon G Patching,Jocelyn M Baldwin,Stephen A Baldwin,Ekaterina Ivanova,David Sharples

doi:10.1099/mic.0.000266

Abstract

This work reports the evolutionary relationships, amplified expression, functional characterization 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 12 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 determined 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 energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3 μM, 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 recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-β-d-maltoside and purified. The isolated protein contained a substantial proportion of α-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 PucI.

Highlights

Allantoin is a naturally occurring compound and a major metabolic intermediate in most living organisms, including bacteria, fungi, plants and animals
A further search based on sequence similarity with PucI from B. subtilis identified homologous putative allantoin permeases in 23 other different species of bacteria
A sequence alignment between PucI from B. subtilis and Mhp1 from M. liquefaciens revealed a significant number of conserved residues with 25.1 % identical and a further 26.9 % highly similar (Fig. S3), confirming a close evolutionary relationship between PucI and Mhp1, and classification of PucI as a NCS-1 family transport protein

Summary

Introduction

Allantoin (or 5-ureidohydantoin) is a naturally occurring compound and a major metabolic intermediate in most living organisms, including bacteria, fungi, plants and animals. The compound name originates from its presence in fluid of the allantois embryonic secretory organ (Inman & Downs, 2007; Arora & Papaioannou, 2012) and it is produced in the degradation pathway of purine nucleobases by action of a urate oxidase (or uricase) enzyme on uric acid (Pizzichini et al, 1996; Xi et al, 2000; Johnson et al, 2009). Primates), is non-functional, so uric acid is the end product of purine catabolism and this is excreted in the urine (Johnson et al, 2009). Purines are used as secondary sources of nitrogen under nutrient-limiting conditions and their degradation ends in formation of the primary nitrogen source ammonia, which can be utilized

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Conclusion