Dynamic elements at both cytoplasmically and extracellularly facing sides of the UapA transporter selectively control the accessibility of substrates to their translocation pathway

In the UapA uric acid–xanthine permease of Aspergillus nidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8–TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1–TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (ΔazgA ΔfcyB ΔuapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10–TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1–TMS2 and TMS8–TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of nonphysiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10–TMS11 loop) with Q113L (TMS1–TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway

Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA

Identification of amino acid residues critical for distinguishing mono- and di-Carboxylate substrates in JEN 1

The knowledge of the mechanisms underlying the transport of carboxylic acids is crucial towards an efficient biological production of carboxylates which have been used for many years in industry namely for the production of biodegradable polymers and as substitute for petroleum-derived chemicals. In Saccharomyces cerevisiae, Jen1p is major monocarboxylate H+ symporter specific primarily for lactate, pyruvate and for acetate (Casal et al, 1999). A phylogenetic tree of ScJen1p homologues (Casal et al, 2008) showed the existence of two main clusters: a Jen1 group of proteins (monocarboxylate transporters) and a Jen2-like proteins (dicarboxylate transporters). In this work, we rationally design, combine and analyse novel mutations in two conserved regions located in TMS5 and TMS11 of Jen1p, which we predicted to affect more dramatically Jen1p specificity. The domain in TMS5 was identified by structure/function studies based on phylogenetic molecular comparisons among Jen1p homologues with different specificities and is critical for distinguishing mono- and di-carboxylate permeases. The conserved aminoacids in TMS11 domain pointed to the importance of this domain that was demonstrated to be involved in substrate binding. We thus identify several residues critical for Jen1p activity, among which some also function as critical specificity determinants for the distinction of mono- from di-carboxylates which constitutes a first step towards the elucidation and genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily (TC#2.A.1.12.2) and a tool for the in silico prediction of the function of Jen1p homologues in other fungi of industrial importance.Fundação para a Ciência e a Tecnologia (FCT) SFRH/BPD/22976/2005 (ISS) and SFRH/BD/61530/2009 (JSP

Modeling, substrate docking, and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter

Background: The purine-cytosine FcyB transporter is a prototype member of the NCS1 family. Results: Using homology modeling, substrate docking, and rational mutational analysis, we identify residues critical for function and specificity. Conclusion: Important aspects concerning the molecular mechanism and evolution of transporter specificity are revealed. Significance: The first systematic approach on structure-function-specificity relationships in a eukaryotic NCS1 member is shown

A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.</p> <p>Results</p> <p>A bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding <it>fdx </it>gene in the <it>Proteobacteria </it>phylum, without occurrence in <it>Archaea </it>and <it>Eukaryota</it>. The transcript was found to be monocistronic in <it>Pseudomonas aeruginosa</it>, and not part of an operon in most bacteria. Only <it>fdx </it>genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in <it>Pseudomonas aeruginosa </it>and in <it>Escherichia coli</it>. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the <it>fdx </it>gene in <it>Pseudomonas aeruginosa </it>abolished growth, unless a plasmid copy of the gene was provided to the deleted strain.</p> <p>Conclusions</p> <p>The gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in <it>Pseudomonas aeruginosa</it>. These data identify a new potential antimicrobial target in this and other pathogenic <it>Proteobacteria</it>.</p

Identification of amino acid residues critical for the substrate translocation in lactate permease JEN1p of saccharomyces cerevisiae

Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate:H+ symporter specific primarily for lactate, pyruvate and for acetate (TC # 2.A.1.12.2) (Casal et al., 1999). A phylogenetic tree of ScJen1p homologues (Casal et al., 2008) showed the existence of two main clusters: a Jen1 group (monocarboxylate transporters) and a Jen2-like (dicarboxylate transporters). Structure-function relationships in Jen1p have been approached by using a rational mutational analysis of conserved amino acid residues (Soares-Silva et al., 2007). Analysis of the conserved sequence 379NXX[S/T]HX[S/T]QDXXXT391, located in transmembrane segment seven (TMS-VII), showed that residues N379, H383 or D387 are necessary for function and specificity, while Q386 is important for the kinetics of Jen1p-mediated transport. In this work, we rationally designed and analyzed novel mutations in conserved regions located in TMS-II, TMS-V and TMS-XI of Jen1p, which we predicted to affect Jen1p specificity (distinction between mono and dicarboxylates) and function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is critical for function. Using a model based on Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI are aligned along the protein pore and substrate docking studies reveal a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function. Overall, our results constitute a first step towards the genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily and a tool for the in silico prediction of the function of Jen1p homologues in other fungi (Soares-Silva et al., 2011).I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

A substrate translocation trajectory in the monocarboxylate/h+ symporter jen1

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387, located in transmembrane segment VII (TMS-VII), is part of the substrate translocation pathway. In this work, we rationally design and analyse novel mutations concerning residues in TMS-V and TMS-XI. Our analysis identifies several residues critical for Jen1p function. Among these, F270 (TMS-V) and Q498 (TMS-XI) function as specificity determinants for the distinction of mono- from di-carboxylates, whereas N501 is irreplaceable for function. Using a novel theoretical model created on the basis of Jen1p similarity with GltP permease, we demonstrate that all polar residues in TMS-VII and TMS-XI, shown previously and herein to be critical for function and/or specificity (N379, H383, D387, Q498, N501), are perfectly aligned in a row along an imaginary axis that lies parallel to a protein pore. The model also predicts that the flexible side-chain of an additional polar residue, R188 in TMSII, faces the pore and subsequent mutational analysis showed that this aminoacid, similar to most polar residues of the pore, is irreplaceable for function. Finally, our model shows that the location of F270 and Q498 could justify their role in substrate specificity. Independent substrate docking approaches reveal a ‘trajectory-like’ displacement of the substrate within the Jen1p pore. In this inward-facing trajectory the flexible side-chain of R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family

L-ascorbic acid (vitamin C) is an essential metabolite in animals and plants due to its role as an enzyme co-factor and antioxidant activity. In most eukaryotic organisms, L-ascorbate is biosynthesized enzymatically, but in several major groups, including the primate suborder Haplorhini, this ability is lost due to gene truncations in the gene coding for L-gulonolactone oxidase. Specific ascorbate transporters (SVCTs) have been characterized only in mammals and shown to be essential for life. These belong to an extensively studied transporter family, called Nucleobase-Ascorbate Transporters (NAT). The prototypic member of this family, and one of the most extensively studied eukaryotic transporters, is UapA, a uric acid-xanthine/H+ symporter in the fungus Aspergillus nidulans. Here, we investigate molecular aspects of NAT substrate specificity and address the evolution of ascorbate transporters apparently from ancestral nucleobase transporters. We present a phylogenetic analysis, identifying a distinct NAT clade that includes all known L-ascorbate transporters. This clade includes homologues only from vertebrates, and has no members in non-vertebrate or microbial eukaryotes, plants or prokaryotes. Additionally, we identify within the substrate-binding site of NATs a differentially conserved motif, which we propose is critical for nucleobase versus ascorbate recognition. This conclusion is supported by the amino acid composition of this motif in distinct phylogenetic clades and mutational analysis in the UapA transporter. Together with evidence obtained herein that UapA can recognize with extremely low affinity L-ascorbate, our results support that ascorbate-specific NATs evolved by optimization of a sub-function of ancestral nucleobase transporters