scholarly journals The functional roles of the three copper sites associated with the methionine-rich insert in the multicopper oxidase CueO fromE. coli

Metallomics ◽  
2015 ◽  
Vol 7 (5) ◽  
pp. 776-785 ◽  
Author(s):  
Laura Cortes ◽  
Anthony G. Wedd ◽  
Zhiguang Xiao
Keyword(s):  
Oxidase Activity ◽  
Multicopper Oxidase ◽  
Functional Roles ◽  
E Coli ◽  
Copper Sites

The methionine-rich insert in the multicopper oxidase CueO fromE. coliaccommodates three copper sites that play distinct but related roles in Cu(i) extraction and oxidation and that induce robust cuprous oxidase activity under physiologically relevant conditions.

scholarly journals Anti-Bacterial Effect of CpG-DNA Involves Enhancement of the Complement Systems

2019 ◽  
Vol 20 (14) ◽  
pp. 3397 ◽  
Author(s):  
Kim ◽  
Park ◽  
Kim ◽  
Gautam ◽  
Akauliya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune System ◽  
Bacterial Infections ◽  
Bacterial Activity ◽  
Complement C3 ◽  
Host Immune System ◽  
Cpg Dna ◽  
Functional Roles ◽  
E Coli ◽  
Bacterial Effect

CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.

scholarly journals Decoding the Functional Roles of Cationic Side Chains of the Major Antimicrobial Region of Human Cathelicidin LL-37

2011 ◽  
Vol 56 (2) ◽  
pp. 845-856 ◽  
Author(s):  
Guangshun Wang ◽  
Raquel F. Epand ◽  
Biswajit Mishra ◽  
Tamara Lushnikova ◽  
Vinai Chittezham Thomas ◽  
...  
Keyword(s):  
Immune Modulation ◽  
Membrane System ◽  
Side Chains ◽  
Cationic Antimicrobial Peptide ◽  
Content Type ◽  
Functional Roles ◽  
E Coli ◽  
Bacterial Aggregation ◽  
Role Mapping ◽  
Cationic Residues

ABSTRACTHuman cathelicidin LL-37 is a critical cationic antimicrobial peptide for host defense against infection, immune modulation, and wound healing. This article elucidates the functional roles of the cationic side chains of the major antimicrobial region of LL-37, corresponding to residues 17 to 32 (designated GF-17). Antimicrobial assays, killing kinetics studies, and vesicle leakage experiments all indicate that a conversion of lysines to arginines affected the ability of the peptide to kill the Gram-positiveStaphylococcus aureusstrain USA300. Alanine scanning experiments show thatS. aureusis less sensitive thanEscherichia colito a single cationic residue mutation of GF-17. Among the five cationic residues, R23 appears to be somewhat important in killingS. aureus. However, R23 and K25 of GF-17 are of prime importance in killing the Gram-negative organismE. coli. In particular, R23 is essential for (i) rapid recognition, (ii) permeation of theE. coliouter membrane, (iii) clustering of anionic lipids in a membrane system mimicking theE. coliinner membrane, and (iv) membrane disruption. Bacterial aggregation (i.e., rapid recognition via charge neutralization) is the first step of the peptide action. Structurally, R23 is located in the interface (i.e., the first action layer), a situation ideal for the interactions listed above. In contrast, residues K18, R19, and R29 are on the hydrophilic surface of the amphipathic helix and play only a secondary role. Mapping of the functional spectrum of cationic residues of GF-17 provides a solid basis for engineering bacterium-specific antimicrobials using this highly potent template.

scholarly journals The reconstitution of oxidase activity in membranes derived from a 5-aminolaevulinic acid-requiring mutant of Escherichia coli

1973 ◽  
Vol 136 (4) ◽  
pp. 877-884 ◽  
Author(s):  
Bruce A. Haddock
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Cytochrome B ◽  
Oxidase Activity ◽  
Nadh Dehydrogenase ◽  
Carbon Sources ◽  
Protoporphyrin Ix ◽  
Side Chain ◽  
E Coli ◽  
Aminolaevulinic Acid

1. The reconstitution of oxidase activity in cell-free extracts of a mutant of Escherichia coli K12Ymel, that require 5-aminolaevulinic acid for growth on non-fermentable carbon sources, is described. 2. The reconstitution is dependent on haematin or a haem extract from a prototrophic strain of E. coli, and the product of the reaction has been identified as NADH-reducible cytochrome b. 3. The requirement for haematin cannot be replaced by four other porphyrins. Coproporphyrin III does not inhibit the haematin-dependent reconstitution, mesoporphyrin IX and protoporphyrin IX apparently compete with haematin for a binding site on the cytochrome apoprotein(s) and deuteroporphyrin IX binds to cytochrome apoprotein(s) and cannot be subsequently replaced by haematin. 4. The properties of electron-transport particles from cell-free extracts of the mutant strain, grown aerobically in the presence or absence of 5-aminolaevulinic acid, are described. In the absence of 5-aminolaevulinic acid no detectable cytochromes are produced, and oxidase activities are lowered but there is no apparent effect on the activities of the NADH dehydrogenase and d-lactate dehydrogenase. 5. The reconstitution of oxidase activity by electron-transport particles from cells grown in the absence of 5-aminolaevulinic acid requires ATP and haematin, and the product of the reaction was identified as NADH-reducible cytochrome b. 6. It is concluded that the cytochrome apoproteins are synthesized and incorporated into the cytoplasmic membrane of E. coli in the absence of haem synthesis. The subsequent reconstitution of functional cytochrome(s) requires protohaem, but the nature of the side chain on the 2 and 4 positions of the porphyrin appears to be important.

scholarly journals Linkage between Catecholate Siderophores and the Multicopper Oxidase CueO in Escherichia coli

2004 ◽  
Vol 186 (17) ◽  
pp. 5826-5833 ◽  
Author(s):  
Gregor Grass ◽  
Keshari Thakali ◽  
Phillip E. Klebba ◽  
Daniel Thieme ◽  
Axel Müller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gas Chromatography ◽  
Multicopper Oxidase ◽  
Copper Resistance ◽  
Acid Oxidation ◽  
Dihydroxybenzoic Acid ◽  
Phenoloxidase Activity ◽  
E Coli ◽  
Gas Chromatography Mass Spectroscopy

ABSTRACT The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Φ(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.

scholarly journals Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

2016 ◽  
Vol 82 (13) ◽  
pp. 3774-3782 ◽  
Author(s):  
Kati Geszvain ◽  
Logan Smesrud ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Oxidase Activity ◽  
Sequence Similarity ◽  
Preliminary Investigation ◽  
Model Organism ◽  
Multicopper Oxidase ◽  
Content Type ◽  
Regulatory Pathways ◽  
Heme Peroxidase ◽  
Oxidase Enzyme

ABSTRACTThe oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacteriumPseudomonas putidaGB-1, two Mn(II) oxidase genes, namedmnxGandmcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity inP. putidaGB-1. A strain from which both of the Mn(II) oxidase genes andfleQwere deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQstrain,P. putidaGB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion offleQunmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes.IMPORTANCEThe identity of the Mn(II) oxidase enzyme inPseudomonas putidaGB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate thatP. putidaGB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes inP. putidaGB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis inPseudomonas aeruginosa. Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

scholarly journals Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6691
Author(s):  
Nikolay B. Pestov ◽  
Daniel V. Kalinovsky ◽  
Tatyana D. Larionova ◽  
Alia Z. Zakirova ◽  
Nikolai N. Modyanov ◽  
...  
Keyword(s):  
Western Blotting ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Lysyl Oxidase ◽  
Polyclonal Antibodies ◽  
Primary Amines ◽  
Oxidase Gene ◽  
E Coli ◽  
Haloterrigena Turkmenica ◽  
Amine Oxidase Activity

Background Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic. Methods LOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. Results Cultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells. Conclusion H. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic. Significance This is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

scholarly journals Two marine GH29 α-L-fucosidases from an uncultured Paraglaciecola sp. specifically hydrolyze fucosyl-N-acetylglucosamine regioisomers

2021 ◽  
Author(s):  
Mikkel Schultz-Johansen ◽  
Peter Stougaard ◽  
Birte Svensson ◽  
David Teze
Keyword(s):  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
Blood Group System ◽  
Substrate Specificities ◽  
Functional Roles ◽  
E Coli ◽  
Terrestrial Environments ◽  
Lewis Blood Group System ◽  
Temperature Optima ◽  
Hydrolase Family

L-Fucose is the most widely distributed L-hexose in marine and terrestrial environments, and presents a variety of functional roles. L-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae, and is found in human milk oligosaccharides and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-L-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in E. coli. All five enzymes (Fp231, Fp239, Fp240, Fp251, Fp284) hydrolyzed the synthetic substrate CNP-α-L-fucose. By screening each of these enzymes against up to 17 fucose-containing oligosaccharides Fp231 and Fp284 showed strict substrate specificities against the fucosyl-N-acetylglucosamine regioisomers Fuc(α1,4)GlcNAc and Fuc(α1,6)GlcNAc, respectively, the former representing a new specificity. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s−1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

scholarly journals Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

The Analyst ◽  
2018 ◽  
Vol 143 (19) ◽  
pp. 4747-4755 ◽  
Author(s):  
Joanna C. Sadler ◽  
Andrew Currin ◽  
Douglas B. Kell
Keyword(s):  
Monoamine Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
Oxidase Activity ◽  
Functional Enrichment ◽  
High Throughput Screen ◽  
Fluorescence Activated Cell Sorting ◽  
E Coli

A novel ultra-high throughput screen forin vivodetection of oxidase activity inE. colicells and its application to directed evolution.

SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

1981 ◽  
Vol 39 ◽  
pp. 448-451
Author(s):  
Susan Chrysegelos ◽  
Kathi Dunn ◽  
Jack Griffith ◽  
Marcia Manning ◽  
Claire Moore
Keyword(s):  
Genome Structure ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
Functional Roles ◽  
E Coli ◽  
Gene 5 Protein ◽  
Nucleoprotein Complex ◽  
Infected Cells ◽  
Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

Heterologous Expression and Application of Multicopper Oxidases from Enterococcus spp. for Degradation of Biogenic Amines

2020 ◽  
Vol 27 ◽  
Author(s):  
Binbin Li ◽  
Yuan Wang ◽  
Linlin Xue ◽  
Shiling Lu
Keyword(s):  
Gene Expression ◽  
Biogenic Amines ◽  
Multicopper Oxidase ◽  
Recombinant Enzyme ◽  
Alkali Resistance ◽  
Prolonged Incubation ◽  
E Coli ◽  
Recombinant Strains ◽  
Enterococcus Spp ◽  
Amine Degradation

Background: Biogenic amines are harmful to human health at a certain extent. As a kind of biogenic amine oxidase, multicopper oxidase can be used to degrade them. Currently, the literature about enzyme from Enterococcus spp. are limited, and recombinant multicopper oxidase might be an effective way to degrade biogenic amines. Objective: (i) Select and identify strains that can degrade biogenic amines, (ii) overexpress enzyme from Enterococcus spp., (iii) measure gene expression and probe amine-degradation differences among strains (native, E. coli DH5α, and L. delbruckii), and (iv) examine the biochemical properties of recombinant multicopper oxidase, (v) apply the recombinant enzyme into smoked horsemeat sausage. Methods: Reverse transcription PCR and high-performance liquid chromatography were performed to examine gene expression and amine degradation rate. Results: The results demonstrated that target enzymes were successfully overexpressed, accompanied by increased aminedegrading activity (P <0.05). Gene from E. faecalis M5B was expressed in L. delbrueckii resulted in degradation rates for phenylethylamine, putrescine, histamine and tyramine of 54%, 52%, 70% and 40%, respectively, significantly higher than achieved by other recombinant strains. Conclusion: In this work, gene expression levels were higher in recombinant M5B than recombinant M2B, regardless of host. E. coli is more stable to express multicopper oxidase. Besides, the amine-degrading ability was markedly increased in the two recombinant strains. After prolonged incubation, the recombinant enzyme could degrade three amines, and it displayed high alkali resistance and thermostability.

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