scholarly journals Investigation of metal binding and activation of Escherichia coli glyoxalase I: kinetic, thermodynamic and mutagenesis studies

2004 ◽  
Vol 377 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Susan L. CLUGSTON ◽  
Rieko YAJIMA ◽  
John F. HONEK
Keyword(s):  
Escherichia Coli ◽  
Isothermal Titration Calorimetry ◽  
Metal Binding ◽  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Titration Calorimetry ◽  
Glyoxalase I ◽  
E Coli ◽  
Catalytic Metal ◽  
Bivalent Metals

GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn2+ but maximal activation with Ni2+. To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the kcat without significantly affecting the Km for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn2+, which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant (Ka)>107 M−1, with the exception of Mn2+ (Ka of the order of 106 M−1). One of the ligands to the catalytic metal, His5, was altered to glutamine, a side chain found in the Zn2+-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn2+-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.

scholarly journals Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

2012 ◽  
Vol 78 (7) ◽  
pp. 2289-2296 ◽  
Author(s):  
Ruth Hall Sedlak ◽  
Marketa Hnilova ◽  
Carolynn Grosh ◽  
Hanson Fong ◽  
Francois Baneyx ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Metal Ion ◽  
Binding Protein ◽  
Metal Resistance ◽  
Transmission Electron Microscopy Analysis ◽  
Binding Peptide ◽  
Content Type ◽  
E Coli

ABSTRACTSilver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerantEscherichia colistrain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of theE. colimaltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strongin vivophenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as thisE. colistrain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactionsin vivo.

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 81-88
Author(s):  
E H Berglin ◽  
M B Edlund ◽  
G K Nyberg ◽  
J Carlsson
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Metal Ion ◽  
Chelating Agent ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Anaerobic Conditions ◽  
Dna Breakage ◽  
E Coli ◽  
K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

scholarly journals Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

1994 ◽  
Vol 300 (2) ◽  
pp. 373-381 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Fluorescence ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

Glyoxalase I – structure, function and a critical role in the enzymatic defence against glycation

2003 ◽  
Vol 31 (6) ◽  
pp. 1343-1348 ◽  
Author(s):  
P.J. Thornalley
Keyword(s):  
Metal Ion ◽  
Catalytic Mechanism ◽  
Critical Role ◽  
Glyoxalase I ◽  
Water Molecules ◽  
Glyoxalase System ◽  
E Coli ◽  
Antimalarial Agents

Glyoxalase I is part of the glyoxalase system present in the cytosol of cells. The glyoxalase system catalyses the conversion of reactive, acyclic α-oxoaldehydes into the corresponding α-hydroxyacids. Glyoxalase I catalyses the isomerization of the hemithioacetal, formed spontaneously from α-oxoaldehyde and GSH, to S-2-hydroxyacylglutathione derivatives [RCOCH(OH)-SG→RCH(OH)CO-SG], and in so doing decreases the steady-state concentrations of physiological α-oxoaldehydes and associated glycation reactions. Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic α-oxoaldehydes. Human glyoxalase I is a dimeric Zn2+ metalloenzyme of molecular mass 42 kDa. Glyoxalase I from Escherichia coli is a Ni2+ metalloenzyme. The crystal structures of human and E. coli glyoxalase I have been determined to 1.7 and 1.5 Å resolution. The Zn2+ site comprises two structurally equivalent residues from each domain – Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules. The Ni2+ binding site comprises His-5A, Glu-56A, His-74B, Glu-122B and two water molecules. The catalytic reaction involves base-catalysed shielded-proton transfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to the thioester product. R- and S-enantiomers of the hemithioacetal are bound in the active site, displacing the water molecules in the metal ion primary co-ordination shell. It has been proposed that Glu-172 is the catalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer; Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product. By analogy with the human enzyme, Glu-56 and Glu-122 may be the bases involved in the catalytic mechanism of E. coli glyoxalase I. The suppression of α-oxoaldehyde-mediated glycation by glyoxalase I is particularly important in diabetes and uraemia, where α-oxoaldehyde concentrations are increased. Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increased glycation and tissue damage. Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to the accumulation of α-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumour and antimalarial agents. Glyoxalase I has a critical role in the prevention of glycation reactions mediated by methylglyoxal, glyoxal and other α-oxoaldehydes in vivo.

Interaction of Humic Acid with Graphene Oxide: Relation to Antibacterial Activities Against Escherichia coli

2021 ◽  
Vol 21 (3) ◽  
pp. 1430-1438
Author(s):  
Hongyang Yu ◽  
Runxuan Chu ◽  
Xue Li ◽  
Bing Wang ◽  
Wei Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Graphene Oxide ◽  
Humic Acid ◽  
Environmental Remediation ◽  
Saline Solution ◽  
Antibacterial Activities ◽  
Titration Calorimetry ◽  
E Coli ◽  
Dose Dependent

Graphene oxide (GO) sheets attracted great attention as effectively antibacterial agents in water treatment and environmental remediation applications. In the study, the interaction of humic acid (HA) as the model of natural organic matter (NOM) with GO and their antibacterial activities against Escherichia coli (E. coli) was investigated. The interaction between GO and HA molecules was analyzed by isothermal titration calorimetry (ITC) and fluorescence spectroscopy analysis. The study demonstrated that GO reaction with HA was a spontaneously exothermic process, which enabled formation of stable and well dispersed GO-HA complex in aqueous solution. Both GO and GO-HA could significantly inhibit the growth of E. coli and present dose-dependent bactericidal property. GO and GO-HA showed more obvious antibacterial activity in saline solution than in LB broth. We suggest the surface wrinkles of GO and GO-HA could contribute to the firm wrapping of E. coli, which is the principle factor for the antibacterial activity of GO and GO-HA. Especially, GO-HA exhibit less surface wrinkles in comparison with GO, corresponding to its reduced antibacterial activity in saline solution.

On-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells

Metallomics ◽  
2015 ◽  
Vol 7 (10) ◽  
pp. 1399-1406 ◽  
Author(s):  
Yuchuan Wang ◽  
Ligang Hu ◽  
Xinming Yang ◽  
Yuen-Yan Chang ◽  
Xuqiao Hu ◽  
...  
Keyword(s):  
Metal Binding ◽  
Inductively Coupled Plasma ◽  
Binding Properties ◽  
E Coli ◽  
Inductively Coupled ◽  
Metal Selectivity ◽  
Icp Ms ◽  
Plasma Mass Spectrometry ◽  
On Line ◽  
Line Coupling

Quantitative analysis of metal selectivity of overexpressed metalloproteins in cells by GE-ICP-MS.

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

Structure–function relationships in Escherichia coli adenylate cyclase

2008 ◽  
Vol 415 (3) ◽  
pp. 449-454 ◽  
Author(s):  
Jürgen U. Linder
Keyword(s):  
Escherichia Coli ◽  
Adenylate Cyclase ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Vibrio Vulnificus ◽  
Class I ◽  
Class Iii ◽  
E Coli ◽  
Metal Cofactor ◽  
Binding Residues

Class I adenylate cyclases are found in γ- and δ-proteobacteria. They play central roles in processes such as catabolite repression in Escherichia coli or development of full virulence in pathogens such as Yersinia enterocolitica and Vibrio vulnificus. The catalytic domain (residues 2–446) of the adenylate cyclase of E. coli was overexpressed and purified. It displayed a Vmax of 665 nmol of cAMP·mg−1·min−1 and a Km of 270 μM. Titration of the metal cofactor Mg2+ against the substrate ATP showed a requirement for free metal ions in addition to the MgATP complex, suggesting a two-metal-ion mechanism as is known for class II and class III adenylate cyclases. Twelve residues which are essential for catalysis were identified by mutagenesis of a total of 20 polar residues conserved in all class I adenylate cyclases. Five essential residues (Ser103, Ser113, Asp114, Asp116 and Trp118) were part of a region which is found in all members of the large DNA polymerase β-like nucleotidyltransferase superfamily. Alignment of the E. coli adenylate cyclase with the crystal structure of a distant member of the superfamily, archaeal tRNA CCA-adding enzyme, suggested that Asp114 and Asp116 are the metal-cofactor-ion-binding residues. The S103A mutant had a 17-fold higher Km than wild-type, demonstrating its important role in substrate binding. In comparison with the tRNA CCA-adding enzyme, Ser103 of the E. coli adenylate cyclase apparently binds the γ-phosphate group of ATP. Consistent with this function, the S103A mutation caused a marked reduction of discrimination between ATP- and ADP- or AMP-derived inhibitors.

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