Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity - PubMed (original) (raw)

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

Pikyee Ma et al. Microbiology (Reading). 2016 May.

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.

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Figures

Fig. 1.

Metabolic pathway for catabolism of purine nucleobases in B. subtilis. The large oval represents the inner cell membrane of B. subtilis containing transport proteins for purine nucleobases and their catabolites (black circles) encoded by the given genes: pbuG (hypoxanthine and guanine), pbuX (xanthine), pucJ/pucK (uric acid), pucI (allantoin) and pbuE (adenine and hypoxanthine efflux). Compound structures are numbered as follows: 1, adenine; 2, hypoxanthine; 3, guanine; 4, xanthine; 5, uric acid; 6, allantoin; 7, allantoic acid; 8, ureidoglycine; 9, ureidoglycolic acid; 10, urea; 11, glyoxylic acid. The genes encoding the enzymes for the conversion reactions are labelled on the reaction arrows. For simplicity, the other reactants and reaction products involved in each conversion are not shown on the diagram, but are listed as follows: adeC (adenine deaminase, H2O → NH3), gde (guanine deaminase, H2O → NH3), pucABCDE (xanthine dehydrogenase, O2 → H2O2), pucLM (uricase, O2 → CO2), pucH (allantoinase, O2 → CO2), pucF (allantoic acid aminohydrolase, H2O → CO2+NH3), pucG (ureidoglycolase). This diagram was constructed based on information given in: Nygaard et al. (1996), (2000); Schultz et al. (2001); Saxild et al. (2001); Beier et al. (2002); Nygaard & Saxild (2005); Goelzer et al. (2008).

Fig. 2.

Induced amplified expression of the PucI(His6) protein in E. coli. (a) Growth curves for BL21(DE3) cells containing the pTTQ18-pucI(His6) construct in LB (squares), 2TY (circles) and M9 minimal (triangles) media supplemented with 20 mM glycerol left uninduced (black shapes) or induced with 0.5 mM IPTG at OD680 0.4–0.6 (white shapes). (b) SDS-PAGE analysis of total membranes prepared from the cells described above. The arrows indicate the position of the amplified PucI(His6) protein. M, Molecular mass markers; I, induced; U, uninduced. (c, d) Effect of induction time on amplified expression of the PucI(His6) protein in E. coli shown by SDS-PAGE (c) and Western blot (d) analysis of total membrane preparations from cultures of BL21(DE3) cells containing the pTTQ18-pucI(His6) construct in M9 minimal medium supplemented with 20 mM glycerol uninduced (left) or induced with 0.5 mM IPTG at OD680 0.4–0.6 (right) and grown for the given further length of time, from 0.1 to 22 h. The arrows indicate the position of the amplified PucI(His6) protein. M, Molecular mass markers.

Fig. 3.

Evolutionary relationships of PucI with NCS-1 family transporters. Phylogenetic tree showing evolutionary relationships of PucI from B. subtilis (P94575) with experimentally characterized bacterial, fungal (Fur-type and Fcy-type) and plant NCS-1 family transport proteins. The NCS-1 proteins are: Mhp1 from M. liquefaciens (D6R8X8), CodB from E. coli (P0AA82), FurA from Asp. nidulans (Q5BFM0), FurD from Asp. nidulans (A6N844), FurE from Asp. nidulans (Q5ATG4), Fur4 from Sac. cerevisiae (P05316), Dal4 from Sac. cerevisiae (Q04895), Fui1 from Sac. cerevisiae (P38196), FcyB from Asp. nidulans (C8V329), Fcy2 from Sac. cerevisiae (P17064), Thi7 from Sac. cerevisiae (Q05998), Tpn1 from Sac. cerevisiae (P53099), Nrt1 from Sac. cerevisiae (Q08485), AtNCS1 (PLUTO) from Ara. thaliana (Q9LZD0), CrNCS1 from C. reinhardtii (A8J166), ZmNCS1 from Z. mays (B4FJ20), SvNCS1 from Set. viridis (V9SBV7). Protein sequences were taken from the UniProt KnowledgeBase (

http://www.uniprot.org

/) and aligned using the online multiple sequence alignment tool

clustal

Omega (

http://www.ebi.ac.uk/Tools/msa/clustalo/

; Sievers et al., 2011). The resultant neighbour-joining phylogenetic tree was exported in Newick format and drawn using the online tool Phylodendron (

http://iubio.bio.indiana.edu/treeapp/treeprint-form.html

). The overall sequence homology (percentage of identical plus highly similar residues) of PucI with Mhp1 and with each group of proteins is given in parentheses. These values were calculated from the sequence alignments shown in Figs S3, S4 and S5.

Fig. 4.

Putative membrane topology of the PucI protein from B. subtilis and conserved residues shared with bacterial putative allantoin permeases and with the hydantoin transport protein Mhp1 from M. liquefaciens. (a, b) The putative membrane topology of the PucI protein (Bsu3645, P94575, ALLP_BACSU) from B. subtilis (strain 168) based on analyses of its sequence using the membrane topology prediction tools

tmhmm

server v. 2.0 (

http://www.cbs.dtu.dk/services/TMHMM/

; Krogh et al., 2001) and

topcons

consensus prediction server (

http://topcons.cbr.su.se/

; Bernsel et al., 2009) (Fig. S6). The putative 12 transmembrane-spanning α-helices are drawn from the N- to the C-terminal end left to right and labelled using roman numerals I–XII. (a) Colours highlight specific types of amino acid residue as follows: positively charged (arginine and lysine, red), negatively charged (aspartic acid and glutamic acid, blue), cysteine (yellow), tryptophan (pink), histidine (orange), proline (green). (b) Grey shading shows residues that are conserved between PucI from B. subtilis and 19 other proteins encoded by allP genes. (c) This diagram is based on an alignment between the sequences for PucI from B. subtilis and the hydantoin transport protein Mhp1 from M. liquefaciens and on the positions of helices in the crystal structure of Mhp1 with bound benzylhydantoin (PDB 4D1B; Simmons et al., 2014) (Fig. S3). Residues are shaded to show those that are conserved between PucI and

Mhp1. In

(b) and (c), identical residues are shown as dark grey and highly similar residues are pale grey. In (c), the red circles highlight residues identical with those in Mhp1 that are involved in direct binding interactions with the hydantoin substrate: Trp117, Gln121, Trp220, Asn318, Leu363.

Fig. 5.

PucI-mediated 14C-allantoin uptake into energized whole cells. (a) Time-course of 14C-allantoin uptake. Uptake of 14C-allantoin (50 μM) at time points over 60 min into energized E. coli BL21(DE3) cells containing the empty plasmid pTTQ18 (squares) or the construct pTTQ18-pucI(His6) (circles) that were uninduced (white shapes) or induced with IPTG (black shapes). (b) Concentration dependence of initial rate PucI-mediated 14C-allantoin uptake. Michaelis–Menten plots for the uptake of 14C-allantoin at concentrations up to 500 μM after 15 s into energized E. coli BL21(DE3) cells containing the construct pTTQ18-pucI(His6) that were uninduced (black circles) or induced with IPTG (black squares). The plot for induced cells was analysed by a least-squares fit to obtain values for the apparent affinity of initial-rate transport (K mapp) and the maximum velocity (V max) of 24.4 ± 3 μM and 14.8 nmol (mg cells)− 1, respectively. For all measurements, cells were cultured in minimal medium with 20 mM glycerol and induced at OD680 0.4–0.6 with 0.5 mM IPTG for 1 h. Harvested cells were washed three times with assay buffer (150 mM KCl, 5 mM MES, pH 6.6) and resuspended to an OD680 of 2.0. Cells were energized with 20 mM glycerol and bubbled air for 3 min, followed by incubation with 14C-allantoin at the given concentrations and removal of aliquots for analysis at the given times. The data points represent the mean of triplicate measurements and the error bars represent

sem

Fig. 6.

Competition of PucI-mediated 14C-allantoin uptake into energized whole cells by unlabelled compounds. Relative uptake (%) of 14C-allantoin (50 μM) after 15 s and 2 min into energized E. coli BL21(DE3) cells containing the construct pTTQ18-pucI(His6) that were induced with IPTG in the presence of a number of unlabelled competing compounds (500 μM). Cells were cultured in minimal medium with 20 mM glycerol and induced at OD680 0.4–0.6 with 0.5 mM IPTG for 1 h. Harvested cells were washed three times with assay buffer (150 mM KCl, 5 mM MES, pH 6.6) and resuspended to an OD680 of 2.0. Cells were energized with 20 mM glycerol and bubbled air for 3 min followed by incubation with 14C-allantoin (50 μM) and removal of aliquots for analysis after 15 s and 2 min. Unlabelled competing compounds (500 μM) were introduced prior to the energization period. The non-competed uptake rate was taken as 100 % corresponding to 14.6 and 30.4 nmol 14C-allantoin (mg cells)− 1 for 15 s and 2 min post addition of 14C-allantoin, respectively. Final uptake samples contain 2 % DMSO or no DMSO for the control sample (denoted by ‘None’ in the figure). The data points represent the mean of duplicate measurements. Structures of many of the compounds are shown in Fig. S9.

Fig. 7.

Ligand binding site in a homology model of PucI. (a) Homology model of PucI (green carbon atoms) docked with (S)-allantoin (PDBeChem 3AL) superimposed with the crystal structure of the Mhp1-benzylhydantoin complex (PDB 4D1B; Simmons et al., 2014; yellow carbon atoms) highlighting key differences in the ligand binding site of PucI. (b) PucI protein interactions with allantoin represented by LigPlot+ (

http://www.ebi.ac.uk/thornton-srv/software/LigPlus/

; Laskowski & Swindells, 2011). Residues are labelled accordingly to the PucI protein.

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Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity - PubMed (original) (raw)