ABSTRACT
With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.
Subject(s)
Anopheles , Guanidines , Insecticides , Mosquito Vectors , Neonicotinoids , Nitro Compounds , Receptors, Nicotinic , Animals , Anopheles/metabolism , Anopheles/genetics , Anopheles/drug effects , Neonicotinoids/pharmacology , Receptors, Nicotinic/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/chemistry , Insecticides/pharmacology , Insecticides/chemistry , Nitro Compounds/pharmacology , Nitro Compounds/chemistry , Guanidines/pharmacology , Mosquito Vectors/drug effects , Mosquito Vectors/genetics , Xenopus laevis , Ligands , Pyridines/pharmacology , Malaria/transmission , Malaria/parasitology , Thiazoles/pharmacology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazines/pharmacology , Thiazines/chemistry , Oocytes/metabolism , Oocytes/drug effects , Female , Insect Proteins/metabolism , Insect Proteins/genetics , Insect Proteins/chemistry , Imidazoles/pharmacology , Imidazoles/chemistryABSTRACT
Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC50) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC50: 12.6-305 µM, MIC: 0.5-128 µg ml-1). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity.
Subject(s)
Anti-Bacterial Agents , Bacillus subtilis , Histidine Kinase , Microbial Sensitivity Tests , Naphthoquinones , Naphthoquinones/pharmacology , Naphthoquinones/chemical synthesis , Naphthoquinones/chemistry , Histidine Kinase/antagonists & inhibitors , Histidine Kinase/metabolism , Bacillus subtilis/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Gene Expression Regulation, Bacterial , QuinonesABSTRACT
The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94â Å resolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.
Subject(s)
Amine Oxidase (Copper-Containing) , Crystallography, X-Ray , Catalysis , Amines , KetonesABSTRACT
Copper amine oxidase from Arthrobacter globiformis (AGAO) catalyses the oxidative deamination of primary amines via a large conformational change of a topaquinone (TPQ) cofactor during the semiquinone formation step. This conformational change of TPQ occurs in the presence of strong hydrogen bonds and neighboring bulky amino acids, especially the conserved Asn381, which restricts TPQ conformational changes over the catalytic cycle. Whether such a semiquinone intermediate is catalytically active or inert has been a matter of debate in copper amine oxidases. Here, we show that the reaction rate of the Asn381Ala mutant decreases 160-fold, and the X-ray crystal structures of the mutant reveals a TPQ-flipped conformation in both the oxidized and reduced states, preceding semiquinone formation. Our hybrid quantum mechanics/molecular mechanics (QM/MM) simulations show that the TPQ conformational change is realized through the sequential steps of the TPQ ring-rotation and slide. We determine that the bulky side chain of Asn381 hinders the undesired TPQ ring-rotation in the oxidized form, favoring the TPQ ring-rotation in reduced TPQ by a further stabilization leading to the TPQ semiquinone form. The acquired conformational flexibility of TPQ semiquinone promotes a high reactivity of Cu(i) to O2, suggesting that the semiquinone form is catalytically active for the subsequent oxidative half-reaction in AGAO. The ingenious molecular mechanism exerted by TPQ to achieve the "state-specific" reaction sheds new light on a drastic environmental transformation around the catalytic center.
ABSTRACT
Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.
Subject(s)
Gene Expression Regulation, Bacterial , Shigella , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Histidine Kinase/genetics , Humans , Shigella/metabolism , Signal Transduction/physiology , Virulence/geneticsABSTRACT
Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.72â Å, respectively [Murakawa et al. (2020 â¸). Proc. Natl Acad. Sci. USA, 117, 10818-10824]. While joint refinement is effective for the determination of the accurate positions of heavy atoms on the basis of the electron density, the structural information on light atoms (hydrogen and deuterium) derived from the neutron diffraction data might be affected by the X-ray data. To unravel the information included in the neutron diffraction data, the structure determination was conducted again using only the neutron diffraction data at 1.72â Å resolution and the results were compared with those obtained in the previous study. Most H and D atoms were identified at essentially the same positions in both the neutron-only and the X-ray/neutron joint refinements. Nevertheless, neutron-only refinement was found to be less effective than joint refinement in providing very accurate heavy-atom coordinates that lead to significant improvement of the neutron scattering length density map, especially for the active-site cofactor. Consequently, it was confirmed that X-ray/neutron joint refinement is crucial for determination of the real chemical structure of the catalytic site of the enzyme.
ABSTRACT
Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15â mm3), seeding the crushed crystals into the enzyme solution and standing for 1â h at an ambient temperature of â¼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5â µm. The microcrystals diffracted X-rays to a resolution beyond 2.0â Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2â Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Arthrobacter/enzymology , Synchrotrons/instrumentation , Amino Acid Sequence , Crystallography, X-Ray , Lasers , Models, Molecular , Protein Conformation , TemperatureABSTRACT
The EvgS/EvgA two-component signal transduction system in Escherichia coli is activated under mildly acidic pH conditions. Upon activation, this system induces the expression of a number of genes that confer acid resistance. The EvgS histidine kinase sensor has a large periplasmic domain that is required for perceiving acidic signals. In addition, we have previously proposed that the cytoplasmic linker region of EvgS is also involved in the activation of this sensor. The cytoplasmic linker region resembles a Per-ARNT-Sim (PAS) domain, which is known to act as a molecular sensor that is responsive to chemical and physical stimuli and regulates the activity of diverse effector domains. Our EvgS/EvgA reporter assays revealed that under EvgS-activating mildly acidic pH conditions, EvgS was activated only during aerobic growth conditions, and not during anaerobic growth. Studies using EvgS mutants revealed that C671A and C683A mutations in the cytoplasmic PAS domain activated EvgS even under anaerobic conditions. Furthermore, among the electron carriers of the electron transport chain, ubiquinone was required for EvgS activation. The present study proposes a model of EvgS activation by oxidation and suggests that the cytoplasmic PAS domain serves as an intermediate redox switch for this sensor.
ABSTRACT
Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least 8 genes including those encoding 3 nonidentical subunits and 3 modifying enzymes. Our heterologous expression study has revealed that the 8 genes are necessary and sufficient for the QHNDH biogenesis.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/biosynthesis , Electrophoresis, Polyacrylamide Gel , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.
Subject(s)
Bacterial Proteins/metabolism , Coenzymes/metabolism , Dipeptides/metabolism , Flavoproteins/metabolism , Indolequinones/metabolism , Mixed Function Oxygenases/metabolism , Paracoccus denitrificans/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalysis , Coenzymes/chemistry , Dipeptides/chemistry , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavoproteins/chemistry , Indolequinones/chemistry , Mixed Function Oxygenases/chemistry , Paracoccus denitrificans/chemistry , Paracoccus denitrificans/genetics , Paracoccus denitrificans/metabolism , Tryptophan/chemistry , Tryptophan/metabolismABSTRACT
Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Bacterial Proteins/chemistry , Quinones/chemistry , Catalytic Domain , Coenzymes/chemistry , Neutron Diffraction , ProtonsABSTRACT
Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.
Subject(s)
Histidine Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quinones/pharmacology , Dose-Response Relationship, Drug , High-Throughput Screening Assays , Histidine Kinase/metabolism , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-ß-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5'-phosphate.
Subject(s)
Carbon-Sulfur Lyases/metabolism , Carboxy-Lyases/metabolism , Enzyme Assays/methods , Homocysteine/blood , Methionine/blood , Pseudomonas putida/enzymology , Streptomyces/enzymology , Amino Acid Metabolism, Inborn Errors/blood , Amino Acid Metabolism, Inborn Errors/diagnosis , Escherichia coli/genetics , Escherichia coli/metabolism , Glycine N-Methyltransferase/blood , Glycine N-Methyltransferase/deficiency , Homocystinuria/blood , Homocystinuria/diagnosis , Humans , Plasmids/genetics , Pseudomonas putida/genetics , Spectrophotometry/methods , Streptomyces/geneticsABSTRACT
The oxidative deamination of biogenic amines, crucial in the metabolism of a wealth of living organisms, is catalyzed by copper amine oxidases (CAOs). In this work, on the ground of accurate molecular modeling, we provide a clear insight into the unique protonation states of the key catalytic aspartate residue Asp298 and the prosthetic group of topaquinone (TPQ) in the CAO of Arthrobacter globiformis (AGAO). This provides both extensions and complementary information to the crystal structure determined by our recent neutron diffraction (ND) experiment. The hybrid quantum mechanics/molecular mechanics (QM/MM) simulations suggest that the ND structure closely resembles a state in which Asp298 is protonated and the TPQ takes an enolate form. The TPQ keto form can coexist in the fully protonated state. The energetic and structural analyses indicate that the active site structure of the AGAO crystal is not a single state but rather a mixture of the different protonation and conformational states identified in this work.
ABSTRACT
In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.
Subject(s)
Aspartic Acid/analysis , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/metabolism , Fluorescent Dyes/analysis , Histidine/analysis , Multienzyme Complexes/metabolism , Signal Transduction/physiology , Aspartic Acid/metabolism , Bacterial Proteins/metabolism , Fluorescent Dyes/metabolism , Histidine/metabolism , Phosphoproteins/metabolism , Phosphorylation/physiology , Trans-Activators/metabolismABSTRACT
In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQamr), which is in equilibrium with a semiquinone radical (TPQsq). The transition from TPQamr to TPQsq is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQamr and TPQsq in noncryocooled crystals of the enzyme from Arthrobacter globiformis and found that the equilibrium shifts more toward TPQsq in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQsq is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQamr, and TPQsq, in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Arthrobacter/enzymology , Dihydroxyphenylalanine/analogs & derivatives , Catalysis , Coenzymes/chemistry , Dihydroxyphenylalanine/chemistry , Molecular Conformation , Temperature , Thermodynamics , X-Ray DiffractionABSTRACT
The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.
Subject(s)
Bacillus subtilis/drug effects , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Histidine Kinase/genetics , Quinones/pharmacology , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/enzymology , Bacterial Proteins/drug effects , Cell Wall/metabolism , Histidine Kinase/drug effects , Inhibitory Concentration 50 , Mutation , Protein Kinases/drug effects , Protein Kinases/genetics , Protein Kinases/metabolismABSTRACT
l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.
Subject(s)
Bacterial Proteins/metabolism , Carboxy-Lyases/metabolism , Recombinant Proteins/metabolism , Streptomyces/enzymology , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Carbon Dioxide/metabolism , Carboxy-Lyases/classification , Carboxy-Lyases/genetics , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Kinetics , Methionine/metabolism , Molecular Weight , Phylogeny , Propylamines/metabolism , Protein Multimerization , Pyridoxal Phosphate/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Spectrophotometry , Streptomyces/genetics , Substrate Specificity , TemperatureABSTRACT
Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Histidine Kinase/antagonists & inhibitors , Histidine Kinase/chemistry , Quinones/pharmacology , Amino Acid Sequence , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Bacterial Outer Membrane Proteins/drug effects , Bacterial Outer Membrane Proteins/genetics , Conserved Sequence , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/drug effects , Escherichia coli Proteins/genetics , Histidine Kinase/genetics , Models, Structural , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/drug effects , Multienzyme Complexes/genetics , Mutagenesis, Site-Directed , PhosphorylationABSTRACT
Sodium taurocholate cotransporting polypeptide (NTCP) was recently discovered as a hepatitis B virus (HBV) receptor, however, the detailed mechanism of HBV entry is not yet fully understood. We investigated the cellular entry pathway of HBV using recombinant HBV surface antigen L protein particles (bio-nanocapsules, BNCs). After the modification of L protein in BNCs with myristoyl group, myristoylated BNCs (Myr-BNCs) were found to bind to NTCP in vitro, and inhibit in vitro HBV infection competitively, suggesting that Myr-BNCs share NTCP-dependent infection machinery with HBV. Nevertheless, the cellular entry rates of Myr-BNCs and plasma-derived HBV surface antigen (HBsAg) particles were the same as those of BNCs in NTCP-overexpressing HepG2 cells. Moreover, the cellular entry of these particles was mainly driven by heparan sulfate proteoglycan-mediated endocytosis regardless of NTCP expression. Taken together, cell-surface NTCP may not be involved in the cellular uptake of HBV, while presumably intracellular NTCP plays a critical role.