The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter of Aspergillus nidulans. We have previously presented evidence showing that a highly conserved signature motif ([Q/E/P]408-N-X-G-X-X-X-X-T-[R/K/G])417 is involved in UapA function. Here, we present a systematic mutational analysis of conserved residues in or close to the signature motif of UapA. We show that even the most conservative substitutions of residues Q408, N409 and G411 modify the kinetics and specificity of UapA, without affecting targeting in the plasma membrane. Q408 substitutions show that this residue determines both substrate binding and transport catalysis, possibly via interactions with position N9 of the imidazole ring of purines. Residue N409 is an irreplaceable residue necessary for transport catalysis, but is not involved in substrate binding. Residue G411 determines, indirectly, both the kinetics (K(m), V) and specificity of UapA, probably due to its particular property to confer local flexibility in the binding site of UapA. In silico predictions and a search in structural databases strongly suggest that the first part of the NAT signature motif of UapA (Q(408)NNG(411)) should form a loop, the structure of which is mostly affected by mutations in G411. Finally, substitutions of residues T416 and R417, despite being much better tolerated, can also affect the kinetics or the specificity of UapA. Our results show that the NAT signature motif defines the function of the UapA purine translocation pathway and strongly suggest that this might occur by determining the interactions of UapA with the imidazole part of purines.     

Identification of the Substrate Recognition and Transport Pathway in a Eukaryotic Member of the Nucleobase-Ascorbate Transporter (NAT) Family

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.     

The first transmembrane segment (TMS1) of UapA contains determinants necessary for expression in the plasma membrane and purine transport

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter in Aspergillus nidulans. Determinants critical for substrate binding and transport lie in a highly conserved signature motif downstream from TMS8 and within TMS12. Here we examine the role of TMS1 in UapA biogenesis and function. First, using a mutational analysis, we studied the role of a short motif (Q85H86), conserved in all NATs. Q85 mutants were cryosensitive, decreasing (Q85L, Q85N, Q85E) or abolishing (Q85T) the capacity for purine transport, without affecting physiological substrate binding or expression in the plasma membrane. All H86 mutants showed nearly normal substrate binding affinities but most (H86A, H86K, H86D) were cryosensitive, a phenotype associated with partial ER retention and/or targeting of UapA in small vacuoles. Only mutant H86N showed nearly wild-type function, suggesting that His or Asn residues might act as H donors in interactions affecting UapA topology. Thus, residues Q85 and H86 seem to affect the flexibility of UapA, in a way that affects either transport catalysis per se (Q85), or expression in the plasma membrane (H86). We then examined the role of a transmembrane Leu Repeat (LR) motif present in TMS1 of UapA, but not in other NATs. Mutations replacing Leu with Ala residues altered differentially the binding affinities of xanthine and uric acid, in a temperature-sensitive manner. This result strongly suggested that the presence of L77, L84 and L91 affects the flexibility of UapA substrate binding site, in a way that is necessary for high affinity uric acid transport. A possible role of the LR motif in intramolecular interactions or in UapA dimerization is discussed.     

The first transmembrane segment (TMS1) of UapA contains determinants necessary for expression in the plasma membrane and purine transport

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H⁺ symporter in Aspergillus nidulans. Determinants critical for substrate binding and transport lie in a highly conserved signature motif downstream from TMS8 and within TMS12. Here we examine the role of TMS1 in UapA biogenesis and function. First, using a mutational analysis, we studied the role of a short motif (Q⁸⁵H⁸⁶), conserved in all NATs. Q85 mutants were cryosensitive, decreasing (Q85L, Q85N, Q85E) or abolishing (Q85T) the capacity for purine transport, without affecting physiological substrate binding or expression in the plasma membrane. All H86 mutants showed nearly normal substrate binding affinities but most (H86A, H86K, H86D) were cryosensitive, a phenotype associated with partial ER retention and/or targeting of UapA in small vacuoles. Only mutant H86N showed nearly wild-type function, suggesting that His or Asn residues might act as H donors in interactions affecting UapA topology. Thus, residues Q85 and H86 seem to affect the flexibility of UapA, in a way that affects either transport catalysis per se (Q⁸⁵), or expression in the plasma membrane (H⁸⁶). We then examined the role of a transmembrane Leu Repeat (LR) motif present in TMS1 of UapA, but not in other NATs. Mutations replacing Leu with Ala residues altered differentially the binding affinities of xanthine and uric acid, in a temperature-sensitive manner. This result strongly suggested that the presence of L⁷⁷, L⁸⁴ and L⁹¹ affects the flexibility of UapA substrate binding site, in a way that is necessary for high affinity uric acid transport. A possible role of the LR motif in intramolecular interactions or in UapA dimerization is discussed.     

Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers

UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.     

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 Aspergillusnidulans, 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 non-physiological 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 of the thyrotropin-releasing hormone-degrading ectoenzyme. similarities and differences with other members of the M1 family of aminopeptidases and thermolysin

Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE) is a TRH-specific peptidase which catalyzes the inactivation of the peptidergic signal substance TRH. As indicated by sequence alignment, TRH-DE and the other members of the M1 family of aminopeptidases have a distinct set of conserved amino acid residues in common. By replacing amino acid residues that are putatively involved in catalysis, we could demonstrate that the enzymatic activities of the mutants E408D, E442D, E464Q, E464D, Y528F, H507R, and H507F are dramatically decreased, essentially due to the changes of V(max). The mutant enzymes E408Q and E442Q are inactive, whereas the specific enzymatic activity of the mutants R488Q, R488A, and Y554F are similar to that of the wild-type enzyme. These data strongly suggest that E408, E442, Y528, and H507 are involved in the catalytic process of TRH-DE while E464 presumably represents the third zinc-coordinating residue and may be equivalent to E166 in thermolysin. In contrast, amino acid residues R488 and Y554 seem not to be involved in the catalytic mechanism of TRH-DE.     

Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues.

In Aspergillus nidulans, purine uptake is mediated by three transporter proteins: UapA, UapC and AzgA. UapA and UapC have partially overlapping functions, are 62% identical and have nearly identical predicted topologies. Their structural similarity is associated with overlapping substrate specificities; UapA is a high-affinity, high-capacity specific xanthine/uric acid transporter. UapC is a low/moderate-capacity general purine transporter. We constructed and characterized UapA/UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutations. The region including residues 378-446 in UapA (336-404 in UapC) has been shown to be critical for purine recognition and transport. Within this region, we identified: (i) one amino acid residue (A404) important for transporter function but probably not for specificity and two residues (E412 and R414) important for UapA function and specificity; and (ii) a sequence, (F/Y/S)X(Q/E/P) NXGXXXXT(K/R/G), which is highly conserved in all homologues of nucleobase transporters from bacteria to man. The UapC/UapA series of chimeras behaves in a linear pattern and leads to an univocal assignment of functional domains while the analysis of the reciprocal and 'sandwich' chimeras revealed unexpected inter-domain interactions. cDNAs coding for transporters including the specificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.     

Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.

Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.     

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.     

Micro‐scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae

Purification of milligram quantities of target proteins is required for structural and biophysical studies. However, mammalian membrane proteins, many of which are important therapeutic targets, are too unstable to be expressed in heterologous hosts and to be solubilized by detergents. One of the most promising ways to overcome these limitations is to stabilize the membrane proteins by generating variants via introduction of truncated flexible regions, fusion partners, and site‐directed mutagenesis. Therefore, an effective screening strategy is a key to obtaining successful protein stabilization. Herein, we report the micro‐scale and high‐throughput screening of stabilized membrane protein variants using Saccharomyces cerevisiae as a host. All steps of the screening, including cultivation and disruption of cells, solubilization of the target protein, and the pretreatment for fluorescence‐detected size exclusion chromatography (FSEC), could be performed in a 96‐well microplate format. We demonstrated that the dispersion among wells was small, enabling detection of a small but important improvement in the protein stability. We also demonstrated that the thermally stable mutants of a human G protein‐coupled receptor could be distinguished based on an increase of the peak height in the FSEC profile, which was well correlated with increased ligand binding activity of the protein. This strategy represents a significant platform for handling numerous mutants, similar to alanine scanning.     

Mitochondrial ATP synthase residue betaarginine-408, which interacts with the inhibitory site of regulatory protein IF1, is essential for the function of the enzyme

Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.     

Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.     

Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes.

UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged amino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic alpha-helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degrees C, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity (K(M) values of 4-10 microM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity (K(M) values of 1.5-15 microM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA.     

Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism

OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP^ND251) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP^ND251, as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.     

Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistance

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein-tyrosine phosphatase.

The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site-directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904-1913]. Two mutants were constructed and expressed that contained the Trp354-->Phe and Trp354-->Ala substitutions. The K(m) of the W354F and W354A mutants were not significantly different from that of the wild-type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200-fold and 480-fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid-deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.     

Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter

In Aspergillus nidulans UapA is a H⁺-driven transporter specific for xanthine, uric acid and several analogues. Here, genetic and physiological evidence is provided showing that allopurinol is a high-affinity, low-capacity, substrate for UapA. Surprisingly however, transport kinetic measurements showed that, uniquely among all recognized UapA substrates, allopurinol is transported by apparent facilitated diffusion and exhibits a paradoxical effect on the transport of physiological substrates. Specifically, excess xanthine or other UapA substrates inhibit allopurinol uptake, as expected, but the presence of excess allopurinol results in a concentration-dependent enhancement of xanthine binding and transport. Flexible docking approaches failed to detect allopurinol binding in the major UapA substrate binding site, which was recently identified by mutational analysis and substrate docking using all other UapA substrates. These results and genetic evidence suggest that the allopurinol translocation pathway is distinct from, but probably overlapping with, that of physiological UapA substrates. Furthermore, although the stimulating effect of allopurinol on xanthine transport could, in principle, be rationalized by a cryptic allopurinol-specific allosteric site, evidence was obtained supporting that accelerated influx of xanthine is triggered through exchange with cytoplasmically accumulated allopurinol. Our results are in line with recently accumulating evidence revealing atypical and complex mechanisms underlying transport systems.