Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

The structural basis of fungal glucuronoyl esterase activity on natural substrates.

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/ß-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.

Laccase activity measurement by FTIR spectral fingerprinting.

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis. We employed FTIR to monitor enzymatic oxidation of monolignols (sinapyl, coniferyl and p-coumaryl alcohol), sinapic acid, and sinapic aldehyde by four different laccases: three fungal laccases from Trametes versicolor, Trametes villosa and Ganoderma lucidum, respectively, and one bacterial laccase from Meiothermus ruber. By coupling the FTIR measurements with Parallel Factor Analysis (PARAFAC) we established a quantitative assay for assessing laccase activity. By combining PARAFAC modelling with Principal Component Analysis we show the usefulness of this technology as a multivariate tool able to compare and distinguish different laccase reaction patterns. We also demonstrate how the FTIR approach can be used to create a reference system for laccase activity comparison based on a relatively low number of measurements. Such a reference system has potential to function as a high-throughput method for comparing reaction pattern similarities and differences between laccases and hereby identify new and interesting enzyme candidates in large sampling pools.

A comparative structural analysis of the surface properties of asco-laccases.

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates.

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

Simulating Substrate Recognition and Oxidation in Laccases: From Description to Design.

To meet the very specific requirements demanded by industry, proteins must be appropriately tailored. Engineering laccases, to improve the oxidation of small molecules, with applications in multiple fields, is, however, a difficult task. Most efforts have concentrated on increasing the redox potential of the enzyme, but in recent work, we have pursued an alternate strategy to engineering these biocatalysts. In particular, we have found that redesigning substrate binding at the T1 pocket, guided by in silico methodologies, to be a more consistent option. In this work, we evaluate the robustness of our computational approach to estimate activity, emphasizing the importance of the binding event in laccase reactivity. Strengths and weaknesses of the protocol are discussed along with its potential for scoring large numbers of protein sequences and thus its significance in protein engineering.

Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters.

Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referred to as E1XXE2R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y, or Q1XXE2Y were able to accumulate peptide, whereas those with E1XXQ2R, Q1XXE2R, or Q1XXQ2Y were unable to accumulate peptide, and Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E2 to R-E1, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E1XXE2R and E1XXQ2R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E2 has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.

New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay.

Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.

The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL.

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, ß-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and ß-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and ß-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus.

The human alanine-serine-cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of d-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter shows a significantly higher sequence identity (35%) than other bacterial homologues. Therefore, this homologue from Gloeobacter violaceus might represent the best bacterial target for structural studies probing the molecular mechanism of Asc-1. We have over-expressed the G. violaceus transporter by auto-induction, and performed purification and biophysical characterization. In addition, growth studies indicate a preference for alanine as nitrogen source in cells expressing the G. violaceus transporter. It was observed that use of the auto-induction method and subsequent optimization of the length of auto-induction was crucial for obtaining high yields and purity of the transporter. The transporter was purified with yields in the range of 0.2-0.4mg per L culture and eluted in a single peak from a size-exclusion column. The circular dichroism spectrum revealed a folded and apparently all-helical protein.

Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides.

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC(50) value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC(50) values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.

Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31.

The crystal structure of alpha-glucosidase MalA from Sulfolobus solfataricus has been determined at 2.5Angstrom resolution. It provides a structural model for enzymes representing the major specificity in glycoside hydrolase family 31 (GH31), including alpha-glucosidases from higher organisms, involved in glycogen degradation and glycoprotein processing. The structure of MalA shows clear differences from the only other structure known from GH31, alpha-xylosidase YicI. MalA and YicI share only 23% sequence identity. Although the two enzymes display a similar domain structure and both form hexamers, their structures differ significantly in quaternary organization: MalA is a dimer of trimers, YicI a trimer of dimers. MalA and YicI also differ in their substrate specificities, as shown by kinetic measurements on model chromogenic substrates. In addition, MalA has a clear preference for maltose (Glc-alpha1,4-Glc), whereas YicI prefers isoprimeverose (Xyl-alpha1,6-Glc). The structural origin of this difference occurs in the -1 subsite where MalA residues Asp251 and Trp284 could interact with OH6 of the substrate. The structure of MalA in complex with beta-octyl-glucopyranoside has been determined. It reveals Arg400, Asp87, Trp284, Met321 and Phe327 as invariant residues forming the +1 subsite in the GH31 alpha-glucosidases. Structural comparisons with other GH families suggest that the GH31 enzymes belong to clan GH-D.

NAC transcription factors: structurally distinct, functionally diverse.

NAC proteins constitute one of the largest families of plant-specific transcription factors, and the family is present in a wide range of land plants. Here, we summarize the biological and molecular functions of the NAC family, paying particular attention to the intricate regulation of NAC protein level and localization, and to the first indications of NAC participation in transcription factor networks. The recent determination of the DNA and protein binding NAC domain structure offers insight into the molecular functions of the protein family. Research into NAC transcription factors has demonstrated the importance of this protein family in the biology of plants and the need for further studies.

Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.

The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by X-ray crystallography to 1.9A resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix-turn-helix motif; instead it reveals a new transcription factor fold consisting of a twisted beta-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

Crystallization and preliminary X-ray analysis of Alicyclobacillus acidocaldarius endoglucanase CelA.

Crystallization of a family 9 beta-1,4-glucanase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius CelA is reported. Thin plates can be obtained by hanging-drop vapour-diffusion crystallization in high concentrations (60%) of MPD. These crystals are unusual in that they do not bind the dye IZIT in the mother liquor and do not appear to dissolve in water after three weeks or in the storage buffer after 2 d. The crystals diffract weakly and the diffraction pattern is compatible with crystal disorder in one direction. After testing several crystals at the ESRF beamlines ID14-1 and ID14-2, a crystal was found which gave ordered diffraction in all directions. A full data set was collected to 3.0 A resolution, which allowed unambiguous determination of the space group as P2(1)2(1)2 and the unit-cell parameters as a = 85, b = 129.7, c = 48.6 A. Initial promising results from molecular-replacement searches are reported.