Asp Residues Research Articles

Structure-function analyses reveal Arabidopsis thaliana HDA7 to be an inactive histone deacetylase

Histone deacetylases (HDACs), responsible for the removal of acetyl groups from histone tails, are important epigenetic factors. They play a critical role in the regulation of gene expression and are significant in the context of plant growth and development. The Rpd3/Hda1 family of HDACs is reported to regulate key biological processes in plants, such as stress response, seed, embryonic, and floral development. Here, we characterized Arabidopsis thaliana HDA7, a Class I, Rpd3/Hda1 family HDAC. SAXS and AUC results show that the recombinantly expressed and purified histone deacetylase domain of AtHDA7 exists as a monomer in solution. Further, the crystal structure showed AtHDA7 to fold into the typical α/β arginase fold, characteristic of Rpd3/Hda1 family HDACs. Sequence analysis revealed that the Asp and His residues of the catalytic ‘XDXH’ motif present in functional Rpd3/Hda1 family HDACs are mutated to Gly and Pro, respectively, in AtHDA7, suggesting that it might be catalytically inactive. The Asp and His residues are important for Zn2+-binding. Not surprisingly, the crystal structure did not have Zn2+ bound in the catalytic pocket, which is essential for the HDAC activity. Further, our in vitro activity assay revealed AtHDA7 to be inactive as an HDAC. A search in the sequence databases suggested that homologs of AtHDA7 are found exclusively in the Brassicaceae family to which Arabidopsis belongs. It is possible that HDA7 descended from HDA6 through whole genome duplication and triplication events during evolution, as suggested in a previous phylogenetic study.

Bacterial β-carbonic anhydrases.

β-Carbonic anhydrases (β-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. β-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. β-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the β-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II β-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.

Remimazolam attenuates myocardial ischemia-reperfusion injury by inhibiting the NF-ĸB pathway of macrophage inflammation

BackgroundInflammation is a major contributing factor in myocardial ischemia/reperfusion (I/R) injury, and targeting macrophage inflammation is an effective strategy for myocardial I/R therapy. Though remimazolam is approved for sedation, induction, and the maintenance of general anesthesia in cardiac surgery, its effect on cardiac function during the perioperative period has not been reported. Therefore, this research aimed to explore the impact of remimazolam on inflammation during myocardial ischemia/reperfusion (I/R) injury. MethodsAn in vivo myocardial I/R mice model and an in vitro macrophage inflammation model were used to confirm remimazolam's cardiac protective effect. In vivo, we used echocardiography, hematoxylin and eosin (HE), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine remimazolam's therapeutic effects on myocardial I/R injury and inflammation. In vitro, we employed enzyme-linked immunosorbent assay (ELISA), Western blot, Real-time Quantitative PCR (qPCR), flow cytometry, and immunofluorescence staining to assess inflammatory responses, especially remimazolam's effects on macrophage polarization after I/R. Furthermore, molecular docking was used to identify its potential binding targets on the inflammatory pathway to explore the mechanism of remimazolam. ResultsRemimazolam exhibited significant anti-myocardial I/R injury activity by inhibiting macrophage-mediated inflammation to reduce myocardial infarction, enhancing cardiac function. In addition, macrophage depletion counteracted improved cardiac function by remimazolam treatment. Mechanistically, the activated NF-ĸB signaling pathway and phosphorylation of p50 and p65 were repressed for anti-inflammatory effect. Consistently, two binding sites on p50 and p65 were identified by molecular docking to affect their phosphorylation of the Ser, Arg, Asp, and His residues, thus regulating NF-κB pathway activity. ConclusionOur results unveil the therapeutic potential of remimazolam against myocardial I/R injury by inhibiting macrophages polarizing into the M1 type, alleviating inflammation.

Promoter DNA recognition by the Enterococcus faecalis global regulator MafR.

When Enterococcus faecalis is exposed to changing environmental conditions, the expression of many genes is regulated at the transcriptional level. We reported previously that the enterococcal MafR protein causes genome-wide changes in the transcriptome. Here we show that MafR activates directly the transcription of the OG1RF_10478 gene, which encodes a hypothetical protein of 111 amino acid residues. We have identified the P10478 promoter and demonstrated that MafR enhances the efficiency of this promoter by binding to a DNA site that contains the -35 element. Moreover, our analysis of the OG1RF_10478 protein AlphaFold model indicates high similarity to 1) structures of EIIB components of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system, and 2) structures of receiver domains that are found in response regulators of two-component signal transduction systems. However, unlike typical EIIB components, OG1RF_10478 lacks a Cys or His residue at the conserved phosphorylation site, and, unlike typical receiver domains, OG1RF_10478 lacks a conserved Asp residue at the position usually required for phosphorylation. Different from EIIB components and receiver domains, OG1RF_10478 contains an insertion between residues 10 and 30 that, according to ColabFold prediction, may serve as a dimerization interface. We propose that OG1RF_10478 could participate in regulatory functions by protein-protein interactions.

Biochemical and hemostatic description of a thrombin-like enzyme TLBro from Bothrops roedingeri snake venom

Objective: The current study’s objective is to characterize a new throm-bin-like enzyme called TLBro that was obtained from Bothrops roedingeris snake from a biochemical and hemostatic perspective.Methodology: One chromatographic step was used to purify it, producing the serine protease TLBro. Molecular mass was estimated by SDS-PAGE to be between reduced and unreduced by 35 kDa. Tryptic peptide sequencing using Swiss Prot provided the complete amino acid sequence. Expasy.org by conducting a search that is limited to Crotalinae snake serine proteases and displaying a high degree of amino acid sequence.Results: Ser (182) is inhibited by phenylmethylsulfonyl fluoride (PMSF), and TLBro demonstrated the presence of Asp (88) residues. It also deduced the positions of His (43) and Ser (182) in the set of three coordinated amino acids in serine proteases. It was discovered that this substrate had high specificity for BANA, Michaelis-Menten behavior with KM 0 point85 mM and Vmax 1 point89 nmoles -NA/L/min, and high stability between temperatures (15 to 70°C) and pHs (2 point0 to 10 point0). According to doses and incubation times, TLBro degraded fibrin preferentially on the B-chain; additionally, its activities were significantly diminished after preincubation with divalent ions (Zn2 and Cd2). When incubated with PMSF, a particular serine protease inhibitor, enzymatic activities and platelet aggregation were inhibited.Conclusion: The findings revealed distinct structural and functional differences between the serine proteases, adding to the information and assisting in the improvement of the structure-function relationship.

Cloning, Sequencing and Expression of Goonch Bagarius bagarius (Hamilton, 1822) Growth Hormone

Abstract Goonch Bagarius bagarius (Hamilton, 1822) is one of the largest and fastest-growing catfish of the Indus River found at Taunsa Barrage, Pakistan. The full-length cDNA of the growth hormone gene (600 bp) was amplified by reverse transcription of mRNA isolated from the pituitary gland of B. bagarius. The full-length growth hormone gene encodes a putative polypeptide of 200 amino acids with a molecular mass of 22.58 kDa. The precursor of B. bagarius growth hormone (GH) is composed of 22 amino acids as a signal peptide and 178 amino acids of a mature peptide. There were six conserved Cys residues in GH protein (20, 71, 135, 173, 190 and 198) that maintain the structural integrity of this protein. One putative N-glycosylation site was present at the 197th amino acid. The total number of positively charged (Arg and Lys) and negatively charged (Asp and Glu) residues is 42 and 31, respectively. The B. bagarius GH gene shows more than 90% sequence homology with other catfishes. The mature protein GH gene was expressed in Escherichia coli using pET-28 expression vector, and the recombinant protein of 19.5 kDa was detected through SDS-PAGE analysis. This study suggests that cloning and expression of B. bagarius GH gene would provide basic information for transgenic studies aimed at a faster growth rate. This recombinant GH may be produced on a large scale to exploit its growth-promoting function in other cultured fishes.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3)

BackgroundIdentification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function.ResultsWe expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6).ConclusionsA novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

OR16-02 Histone ADP-Ribosylation Site Mutations As Oncogenic Drivers In Breast And Ovarian Cancers

Abstract Disclosure: D. Huang: None. C.V. Camacho: None. A. Nagari: None. W.L. Kraus: Other; Self; - Founder and stockholder for Ribon Therapeutics, Inc. - Founder, stockholder, scientific advisory board member, member of the board of directors for ARase Therapeutics, Inc. - Coholder of U.S. Patent. Recent studies have identified oncohistones, which are encoded by histone genes that harbor missense mutations, as a new class of cancer drivers. We found that many of the oncohistone mutations identified with the highest frequencies in human cancers occur on glutamate (Glu, E) and aspartate (Asp, D) residues, which are potential sites of ADP-ribosylation (ADPRylation). ADPRylation is a post-translational modification, which is catalyzed by the PARP family of ADP-ribosyl transferases and results in the covalent attachment of ADP-ribose on protein substrates. Unlike other well characterized histone modifications, the functions of histone ADPRylation are poorly characterized. Mass spectrometry analyses confirmed that 20 Glu or Asp residues subjected to oncohistone mutation are bona fide sites of ADPRylation by PARP-1. Ectopic expression of each of six different oncohistone mutants from three different core histones (H2B, H3, and H4) altered the proliferation of at least 2 out of 3 cancer cell lines, including MCF-7 and MDA-MB-231 breast cancer cells, as well as OVCAR-3 ovarian cancer cells. We focused on H2B-D51, whose mutation resulted in enhanced cell proliferation in both of the breast cancer cell lines for further characterization of its potential transcription regulatory effects. Proteomic (histone PTMs) and genomic (ATAC-Seq, ChIP-Seq and RNA-Seq) analyses revealed that loss of ADPRylation on H2B-D51 by mutation to alanine (Ala, A) or asparagine (Asn, N) dramatically enhanced p300-mediated acetylation of H2B, altered chromatin accessibility, and changed the pattern of gene expression in MDA-MB-231 cells. To determine the effects of H2B-D51 ADPRylation on cancer cell growth in vivo and their responses to the FDA-approved PARP inhibitor Niraparib, we performed xenograft tumor growth assays in immunocompromised mice using MDA-MB-231 cells ectopically expressing wild-type or D51A mutant histone H2B. Ectopic expression of the H2B-D51A mutant led to a significant increase in tumor growth versus wild-type H2B. Interestingly, these effects were inhibited by treatment with Niraparib, suggesting that the H2B-D51A mutant alone does not confer resistance to PARP inhibitor. Taken together, this study demonstrates that functional sites of Glu/Asp ADPRylation on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of ADPRylation. (This work was supported by grants from the NIH/NIDDK and CPRIT, and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K.) Presentation: Friday, June 16, 2023

Production and characterization of novel thermostable CotA-laccase from Bacillus altitudinis SL7 and its application for lignin degradation

Laccases are multi-copper oxidases and found in ligninolytic bacteria catalyzing the oxidation of both phenolic and non-phenolic compounds, however its application in lignin degradation suffers due to low oxidation rate, which have intensified the search for new laccases. In the present study, spore coat A protein (CotA) encoding gene having laccase like activity from Bacillus altitudinis SL7 (CotA-SL7) was cloned and expressed in Escherichia coli. The purified CotA-SL7 was active at wide range of temperature and pH with optimum activity at 55 °C and pH 5.0. The kinetic parameters of CotA-SL7 was determined with Km, Vmax, and kcat values 0.4 mM, 2777 μmol/min/mg, and 5194 s−1, respectively. Molecular docking revealed the presence of Pro, Phe, Asp, Asn, His, and Ile residues at the active site taking part in the oxidation of ABTS. The purified CotA-SL7 reduced lignin contents by 31 % and changes in lignin structure were analyzed through fourier transformed infrared spectroscopy (FTIR), scanning electron microsscopy (SEM) and gas chromatography mass-spectrometry (GC-MS). The appearance of low molecular size compounds clearly indicates the cleavage of lignin polymer and opening of the benzene ring by purified CotA-SL7. Thus, high catalytic efficiency of CotA-SL7 makes it a suitable bio-catalyst for remediation of lignin contaminated wastewater from pulp and paper industries with clear insights into lignin degradation at molecular level.

Signal peptide and N-glycosylation of N-terminal-CD2v determine the hemadsorption of African swine fever virus.

African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines.

PP2C phosphatases Ptc1 and Ptc2 dephosphorylate PGK1 to regulate autophagy and aflatoxin synthesis in the pathogenic fungus Aspergillus flavus.

Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

The Role of Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aβ42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-β peptide (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aβ42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aβ42 oligomers (AβO). Far-UV CD indicated a shift in the conformational feature of AβOs from the random coil to β-sheet in the presence of PIMT. Binding of bis-ANS to different AβOs (obtained using different concentrations of Aβ42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AβO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AβO with the addition of PIMT, a contrasting role to that on Aβ fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AβO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AβO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aβ42 oligomers and fibrils.

Collision-Induced Dissociation of Citrullinated Peptide Anions.

Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH3/z, c, c-NH3/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages N-terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage N-terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.

Functional and structural identification of iron-binding proteins on tomato (Solanum lycopersicum L.) proteome via in silico approaches

Iron-plant interactions have crucial roles in crop production growth and development. In this study, we have analyzed the whole proteome of tomato (Solanum lycopersicum L.) plants for iron-binding proteins. A total of 213 iron-binding protein candidates were identified in the study. Out of these 213 proteins, 45 were selected for modeling and validated with a high confidence level by using different computational analyses. Results showed that Glu, Cys, Asp, and His amino acid residues were indicators of iron-binding proteins. Besides, mechanistic insights of iron-binding proteins were analyzed by molecular dynamics simulations. Simulation results proved the conformational stabilization of proteins. Validated proteins were further analyzed for subcellular localization, clustered for molecular functions and biological processes. According to the results, iron-binding proteins were mostly located in the chloroplast. Also, these proteins are involved in different molecular and biological roles ranging from oxidation-reduction processes and electron transport chain to protein repair mechanisms. This report provides structural and functional properties of iron-binding proteins for tomato proteome. The study may assist in future research on plant physiology, protein engineering, or bioengineering.

Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects β-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases

Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.2 Å cryoEM structure of qNOR from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. This high-resolution structure provides insight into electron, substrate, and proton pathways, and provides evidence that the quinol binding site not only contains the conserved His and Asp residues but also possesses a critical Arg (Arg720) observed in cytochrome bo3, a respiratory quinol oxidase.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.