Biochemical characterization and gene structure analysis of the 24-kDa glutathione transferase sigma from Taenia solium.

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.

Unveiling success determinants for AMB-assisted phase expansion of fusion proteins in ARP/wARP.

Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.

Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant Mycobacterium tuberculosis clinical isolate.

Mycobacterium tuberculosis catalase-peroxidase (Mt-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical M. tuberculosis isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG). The rate of IN-NAD formation mediated by WT-KatG was 23% greater than L333V-KatG when INH concentration is varied. In contrast to WT-KatG, the crystal structure of the L333V-KatG variant has a perhydroxy modification of the indole nitrogen of W107 from MYW adduct. L333V-KatG shows most of the active site residues in a similar position to WT-KatG; only R418 is in the R-conformation instead of the double R and Y conformation present in WT-KatG. L333V-KatG shows a small displacement respect to WT-KatG in the helix from R385 to L404 towards the mutation site, an increase in length of the coordination bond between H270 and heme Fe, and a longer H-bond between proximal D381 and W321, compared to WT-KatG; these small displacements could explain the altered redox potential of the heme, and result in a less active and stable enzyme.

A Putative New Role of Tv-PSP1 Recognizes IRE and ERE Hairpin Structures from Trichomonas vaginalis.

To understand whether protein Tv-PSP1 from Trichomonas vaginalis recognizes mRNA parasite stem-loop structures, we conducted REMSA and intrinsic fluorescence assays. We found the recombinant Tv-PSP1 structure, determined with X-ray crystallography, showed unusual thermal stability of the quaternary structure, associated with a disulfide bridge CYS76-CYS104. To gain deeper insight into the Tv-PSP1 interaction with mRNA stem-loops (mRNAsl) and its relationship with thermal stability, we also used an integrated computational protocol that combined molecular dynamics simulations, docking assays, and binding energy calculations. Docking models allowed us to determine a putative contact surface interaction region between Tv-PSP1 and mRNAsl. We determined the contributions of these complexes to the binding free energy (ΔGb) in the electrostatic (ΔGelec) and nonelectrostatic (ΔGnon-elec) components using the Adaptive Poisson-Boltzmann Solver (APBS) program. We are the first, to the best of our knowledge, to show the interaction between Tv-PSP1 and the stem-loop structures of mRNA.

PCNA from Thermococcus gammatolerans: A protein involved in chromosomal DNA metabolism intrinsically resistant at high levels of ionizing radiation.

Proliferating cell nuclear antigen (PCNA) is an essential protein for cell viability in archaea and eukarya, since it is involved in DNA replication and repair. In order to obtain insights regarding the characteristics that confer radioresistance, the structural study of the PCNA from Thermococcus gammatolerans (PCNATg ) in a gradient of ionizing radiation by X-ray crystallography was carried out, together with a bioinformatic analysis of homotrimeric PCNA structures, their sequences, and their molecular interactions. The results obtained from the datasets and the accumulated radiation dose for the last collection from three crystals revealed moderate and localized damage, since even with the loss of resolution, the electron density map corresponding to the last collection allowed to build the whole structure. Attempting to understand this behavior, multiple sequence alignments, and structural superpositions were performed, revealing that PCNA is a protein with a poorly conserved sequence, but with a highly conserved structure. The PCNATg presented the highest percentage of charged residues, mostly negatively charged, with a proportion of glutamate more than double aspartate, lack of cysteines and tryptophan, besides a high number of salt bridges. The structural study by X-ray crystallography reveals that the PCNATg has the intrinsic ability to resist high levels of ionizing radiation, and the bioinformatic analysis suggests that molecular evolution selected a particular composition of amino acid residues, and their consequent network of synergistic interactions for extreme conditions, as a collateral effect, conferring radioresistance to a protein involved in the chromosomal DNA metabolism of a radioresistant microorganism.

The molecular basis of the nonprocessive elongation mechanism in levansucrases.

Levansucrases (LSs) synthesize levan, a ß2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (-1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wildtype (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

Inductive effects in amino acids and peptides: Ionization constants and tryptophan fluorescence.

Although inductive effects in organic compounds are known to influence chemical properties such as ionization constants, their specific contribution to the properties/behavior of amino acids and functional groups in peptides remains largely unexplored. In this study we developed a computationally economical algorithm for ab initio calculation of the magnitude of inductive effects for non-aromatic molecules. The value obtained by the algorithm is called the Inductive Index and we observed a high correlation (R2 = 0.9427) between our calculations and the pKa values of the alpha-amino groups of amino acids with non-aromatic side-chains. Using a series of modified amino acids, we also found similarly high correlations (R2 > 0.9600) between Inductive Indexes and two wholly independent chemical properties: i) the pKa values of ionizable side-chains and, ii) the fluorescence response of the indole group of tryptophan. After assessing the applicability of the method of calculation at the amino acid level, we extended our study to tryptophan-containing peptides and established that inductive contributions of neighboring side-chains are transmitted through peptide bonds. We discuss possible contributions to the study of proteins.

Characterization of a Novel Functional Trimeric Catechol 1,2-Dioxygenase From a Pseudomonas stutzeri Isolated From the Gulf of Mexico.

Catechol 1,2 dioxygenases (C12DOs) have been studied for its ability to cleavage the benzene ring of catechol, the main intermediate in the degradation of aromatic compounds derived from aerobic degradation of hydrocarbons. Here we report the genome sequence of the marine bacterium Pseudomonas stutzeri GOM2, isolated from the southwestern Gulf of Mexico, and the biochemical characterization of its C12DO (PsC12DO). The catA gene, encoding PsC12DO of 312 amino acid residues, was cloned and expressed in Escherichia coli. Many C12DOs have been described as dimeric enzymes including those present in Pseudomonas species. The purified PsC12DO enzyme was found as an active trimer, with a molecular mass of 107 kDa. Increasing NaCl concentration in the enzyme reaction gradually reduced activity; in high salt concentrations (0.7 M NaCl) quaternary structural analysis determined that the enzyme changes to a dimeric arrangement and causes a 51% decrease in specific activity on catechol substrate. In comparison with other C12DOs, our enzyme showed a broad range of action for PsC12DO in solutions with pH values ranging from neutral to alkaline (70%). The enzyme is still active after incubation at 50°C for 30 min and in low temperatures to long term storage after 6 weeks at 4°C (61%). EDTA or Ca2+ inhibitors cause no drastic changes on residual activity; nevertheless, the activity of the enzyme was affected by metal ions Fe3+, Zn2+ and was completely inhibited by Hg2+. Under optimal conditions the k cat and K m values were 16.13 s-1 and 13.2 µM, respectively. To our knowledge, this is the first report describing the characterization of a marine C12DOs from P. stutzeri isolated from the Gulf of Mexico that is active in a trimeric state. We consider that our enzyme has important features to be used in environments in presence of EDTA, metals and salinity conditions.

Implications of the mutation S164A on Bacillus subtilis levansucrase product specificity and insights into protein interactions acting upon levan synthesis.

Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB, leading to the enhanced synthesis of medium and high molecular weight polymer and a blasto-oligosaccharide series with a polymerization degree of 2-10. A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose. Despite its modified product specificity, protein-carbohydrate and protein-protein interactions are still a major factor affecting size and distribution of levan molecular weight. This study highlights the importance of critical factors such as protein concentration in the analysis of wild-type and mutagenized levansucrases. Docking experiments with the crystal structures of SacB and variant S164A - the latter obtained at a 2.6 Å resolution - identified unreported potential binding subsites for fructosyl moieties on the surface of both enzymes.

The crystal structure of ESBL TLA-1 in complex with clavulanic acid reveals a second acylation site.

ß-lactamases are the main molecules responsible for giving bacterial resistance against ß-lactam antibiotics. The study of ß-lactamases has allowed the development of antibiotics capable of inhibiting these enzymes. In this context, extended spectrum ß-lactamase (ESBL) TLA-1 has spread in Escherichia coli and Enterobacter cloacae clinical isolates during the last 30 years in Mexico. In this research, the 3D structures of ESBL TLA-1 and TLA-1 S70G mutant, both ligand-free and in complex with clavulanic acid were determined by X-ray crystallography. Four clavulanic acid molecules were found in the structure of TLA-1, two of those were intermediaries of the acylation process and were localized covalently bound to two different amino acid residues, Ser70 and Ser237. The coordinates of TLA-1 in complex with clavulanic acid shows the existence of a second acylation site, additional to Ser70, which might be extendable to several members of the subclass A ß-lactamases family. This is the first time that two serines involved in binding clavulanic acid has been reported and described to an atomic level.

Crystal structure of 6aJL2-R24G light chain variable domain: Does crystal packing explain amyloid fibril formation?

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Šresolution by molecular replacement. Crystal belongs to space group I212121 (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo. The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro. In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro, is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.

Transcriptomic and Proteomic Analysis of the Tentacles and Mucus of Anthopleura dowii Verrill, 1869.

Sea anemone venom contains a complex and diverse arsenal of peptides and proteins of pharmacological and biotechnological interest, however, only venom from a few species has been explored from a global perspective to date. In the present study, we identified the polypeptides present in the venom of the sea anemone Anthopleura dowii Verrill, 1869 through a transcriptomic and proteomic analysis of the tentacles and the proteomic profile of the secreted mucus. In our transcriptomic results, we identified 261 polypeptides related to or predicted to be secreted in the venom, including proteases, neurotoxins that could act as either potassium (K+) or sodium (Na+) channels inhibitors, protease inhibitors, phospholipases A2, and other polypeptides. Our proteomic data allowed the identification of 156 polypeptides-48 exclusively identified in the mucus, 20 in the tentacles, and 88 in both protein samples. Only 23 polypeptides identified by tandem mass spectrometry (MS/MS) were related to the venom and 21 exclusively identified in the mucus, most corresponding to neurotoxins and hydrolases. Our data contribute to the knowledge of evolutionary and venomic analyses of cnidarians, particularly of sea anemones.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry.

BACKGROUND: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. METHODS: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. RESULTS: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

X-ray driven reduction of Cpd I of Catalase-3 from N. crassa reveals differential sensitivity of active sites and formation of ferrous state.

Catalases are biotechnologically relevant enzymes because of their applications in food technology, bioremediation, and biomedicine. The dismutation of hydrogen peroxide occurs in two steps; in the first one, the enzyme forms an oxidized compound I (Cpd I) and in the second one, the enzyme is reduced to the ferric state. In this research work, we analyzed the reduction of Cpd I by X-ray radiation damage during diffraction experiments in crystals of CAT-3, a Large-Size Subunit Catalase (LSC) from Neurospora crassa. A Multi-Crystal Data collection Strategy was applied in order to obtain the Cpd I structure at a resolution of 2.2 Å; this intermediate was highly sensitive to X-ray and was easily reduced at very low deposited radiation dose, causing breakage of the Fe=O bond. The comparison of the structures showed reduced intermediates and also evidenced the differential sensitivity per monomer. The resting ferric state was reduced to the ferrous state, an intermediate without a previous report in LSC. The chemically obtained Cpd I and the X-ray reduced intermediates were identified by UV-visible microspectrometry coupled to data collection. The differential sensitivity and the formation of a ferrous state are discussed, emphasizing the importance of the correct interpretation in the oxidation state of the iron heme.

The structure of (E)-biformene synthase provides insights into the biosynthesis of bacterial bicyclic labdane-related diterpenoids.

The labdane-related diterpenoids (LRDs) are a large group of natural products with a broad range of biological activities. They are synthesized through two consecutive reactions catalyzed by class II and I diterpene synthases (DTSs). The structural complexity of LRDs mainly depends on the catalytic activity of class I DTSs, which catalyze the formation of bicyclic to pentacyclic LRDs, using as a substrate the catalytic product of class II DTSs. To date, the structural and mechanistic details for the biosynthesis of bicyclic LRDs skeletons catalyzed by class I DTSs remain unclear. This work presents the first X-ray crystal structure of an (E)-biformene synthase, LrdC, from the soil bacterium Streptomyces sp. strain K155. LrdC was identified as a part of an LRD cluster of five genes and was found to be a class I DTS that catalyzes the Mg2+-dependent synthesis of bicyclic LRD (E)-biformene by the dephosphorylation and rearrangement of normal copalyl pyrophosphate (CPP). Structural analysis of LrdC coupled with docking studies suggests that Phe189 prevents cyclization beyond the bicyclic LRD product through a strong stabilization of the allylic carbocation intermediate, while Tyr317 functions as a general base catalyst to deprotonate the CPP substrate. Structural comparisons of LrdC with homology models of bacterial bicyclic LRD-forming enzymes (CldD, RmnD and SclSS), as well as with the crystallographic structure of bacterial tetracyclic LRD ent-kaurene synthase (BjKS), provide further structural insights into the biosynthesis of bacterial LRD natural products.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)

Trichomonas vaginalis metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme.

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.

The Hcp-like protein HilE inhibits homodimerization and DNA binding of the virulence-associated transcriptional regulator HilD in Salmonella.

HilD is an AraC-like transcriptional regulator that plays a central role in Salmonella virulence. HilD controls the expression of the genes within the Salmonella pathogenicity island 1 (SPI-1) and of several genes located outside SPI-1, which are mainly required for Salmonella invasion of host cells. The expression, amount, and activity of HilD are tightly controlled by the activities of several factors. The HilE protein represses the expression of the SPI-1 genes through its interaction with HilD; however, the mechanism by which HilE affects HilD is unknown. In this study, we used genetic and biochemical assays revealing how HilE controls the transcriptional activity of HilD. We found that HilD needs to assemble in homodimers to induce expression of its target genes. Our results further indicated that HilE individually interacts with each the central and the C-terminal HilD regions, mediating dimerization and DNA binding, respectively. We also observed that these interactions consistently inhibit HilD dimerization and DNA binding. Interestingly, a computational analysis revealed that HilE shares sequence and structural similarities with Hcp proteins, which act as structural components of type 6 secretion systems in Gram-negative bacteria. In conclusion, our results uncover the molecular mechanism by which the Hcp-like protein HilE controls dimerization and DNA binding of the virulence-promoting transcriptional regulator HilD. Our findings may indicate that HilE's activity represents a functional adaptation during the evolution of Salmonella pathogenicity.

Zo-peroxidase: Crystal structure and sequence of a highly-glycosylated peroxidase resistant to high concentrations of H2O2 from Japanese radish.

Understanding Peroxidase (PRXs) enzymatic diversity and functional significance from a three-dimensional point of view is a key point for structural and mechanistic studies. In this context, Zo-peroxidase (ZoPrx) a member of the class III peroxidases and secreted by plants, differs from all previously described PRXs because of its remarkable catalytic stability in the presence of hydrogen peroxide. In this work, we present the crystallographic structure of ZoPrx isolated from Japanese radish, at 2.05 Å resolution. The mature enzyme consists of a single monomer of 308 residues exhibiting the same fold as all previously described members of the plant PRXs superfamily. Furthermore, the enzyme contains a heme b group as the prosthetic group and two Ca2+ binding sites. Moreover, seven N-glycosylation sites were found in the structure, and 49 glycans bound to the two ZoPrx molecules found in the asymmetric unit are clearly visible in the electron density map. The comparison of ZoPrx coordinates with homologous enzymes revealed minor structural changes, in which the residue 177 appears to be responsible for enlarging the access to the heme cavity, the only structural finding which may be related to the H2O2 tolerance of ZoPrx and detected by X-ray crystallography. Because of its characteristics, ZoPrx has a broad range of potential applications from chemical synthesis to environmental biocatalysis, thus its aminoacidic sequence, partially completed using the electron density, and the three-dimensional structure itself, become a possible starting point to engineering heme-peroxidases to enhance oxidative stability.

Structure, kinetics, molecular and redox properties of a cytosolic and developmentally regulated fungal catalase-peroxidase.

CAT-2, a cytosolic catalase-peroxidase (CP) from Neurospora crassa, which is induced during asexual spore formation, was heterologously expressed and characterized. CAT-2 had the Met-Tyr-Trp (M-Y-W) adduct required for catalase activity. Its KM for H2O2 was micromolar for peroxidase and millimolar for catalase activity. A Em = -158 mV reduction potential value was obtained and the Soret band shift suggested a mixture of low and high spin ferric iron. CAT-2 EPR spectrum at 10 K indicated an axial and a rhombic component. With peroxyacetic acid (PAA), formation of Compound I* was observed with EPR. CAT-2 homodimer crystallographic structure contained two K+ ions; Glu107 residues were displaced to bind them. CAT-2 showed the essential amino acid residues for activity in similar positions to other CPs. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. A perhydroxy modification of the indole nitrogen of Trp90 was oriented toward the catalytic His91. In contrast to cytochrome c peroxidase and ascorbate peroxidase, the catalase-peroxidase heme propionates are not exposed to the solvent. Together with other N. crassa enzymes that utilize H2O2 as a substrate, CAT-2 has many tryptophan and proline residues at its surface, probably related to H2O2 selection in water.