scholarly journals Anion Inhibition Studies of the Beta-Carbonic Anhydrase from Escherichia coli

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2564 ◽  
Author(s):  
Sonia Del Prete ◽  
Viviana De Luca ◽  
Alessio Nocentini ◽  
Andrea Scaloni ◽  
Margaret D. Mastrolorenzo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phenylboronic Acid ◽  
Co2 Concentration ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Carbonic Anhydrases ◽  
Gene Encoding ◽  
Beta Carbonic Anhydrase ◽  
Lower Intestine ◽  
Operon Gene

The interconversion of CO2 and HCO3− is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3−. In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a β-CA, and CynS gene, encoding for the cyanase. CynT (β-CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second β-CA (CynT2 or Can or yadF), as well as a γ and ι-CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: kcat = 5.3 × 105 s−1 and kcat/KM = of 4.1 × 107 M−1 s−1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 µM). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

scholarly journals Cloning and Sequencing of a Poly(dl-Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli

2003 ◽  
Vol 69 (5) ◽  
pp. 2498-2504 ◽  
Author(s):  
Yukie Akutsu-Shigeno ◽  
Teerawat Teeraphatpornchai ◽  
Kamonluck Teamtisong ◽  
Nobuhiko Nomura ◽  
Hiroo Uchiyama ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Amino Acid ◽  
Functional Expression ◽  
Amino Acid Residues ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
Butylene Succinate ◽  
Expression In Escherichia Coli ◽  
Poly Ethylene

ABSTRACT The gene encoding a poly(dl-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(ε-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

scholarly journals Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli

1988 ◽  
Vol 256 (3) ◽  
pp. 741-749 ◽  
Author(s):  
N Allison ◽  
C H Williams ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Control ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Lambda Repressor ◽  
Intact Gene ◽  
In The Wild ◽  
Lipoamide Dehydrogenase

A ‘split-gene’ technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor. After thermo-induction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188→Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.

scholarly journals Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12

1988 ◽  
Vol 255 (1) ◽  
pp. 35-43 ◽  
Author(s):  
S C Andrews ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Evolutionary Relationship ◽  
Cysteine Residue ◽  
Start Codon ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Imp Dehydrogenase ◽  
Escherichia Coli K12 ◽  
Translational Start

(1) The nucleotide sequence of a 1991 bp segment of DNA that expresses the GMP reductase (guaC) gene of Escherichia coli K12 was determined. (2) This gene comprises 1038 bp, 346 codons (including the initiation codon but excluding the termination codon), and it encodes a polypeptide of Mr 37,437 which is in good agreement with previous maxicell studies. (3) The sequence contains a putative promoter 102 bp upstream of the translational start codon, and this is immediately followed by a (G + C)-rich discriminator sequence suggesting that guaC expression may be under stringent control (4) The GMP reductase exhibits a high degree of sequence identity (34%) with IMP dehydrogenase (the guaB gene product) indicative of a close evolutionary relationship between the salvage pathway and the biosynthetic enzymes, GMP reductase and IMP dehydrogenase, respectively. (5) A single conserved cysteine residue, possibly involved in IMP binding to IMP dehydrogenase, was located within a region that possesses some of the features of a nucleotide binding site. (6) The IMP dehydrogenase polypeptide contains an internal segment of 123 amino acid residues that has no counterpart in GMP reductase and may represent an independent folding domain flanked by (alanine + glycine)-rich interdomain linkers.

scholarly journals Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

2001 ◽  
Vol 183 (15) ◽  
pp. 4543-4550 ◽  
Author(s):  
Mónica A. Delgado ◽  
Marı́a R. Rintoul ◽  
Ricardo N. Farı́as ◽  
Raúl A. Salomón
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Cyclic Peptide ◽  
Nucleotide Sequencing ◽  
Peptide Antibiotic ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Microcin J25

ABSTRACT Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in therpoC gene, encoding the β′ subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-typerpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoCgene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

Insoluble but enzymatically active α-amylase from Bacillus licheniformis

Biologia ◽  
2009 ◽  
Vol 64 (4) ◽  
Author(s):  
Naeem Rashid ◽  
Alia Farooq ◽  
Ikram-ul-Haq ◽  
Muhammad Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Specific Activity ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Mature Protein ◽  
Gene Encoding ◽  
T7 Promoter ◽  
Highly Active

AbstractThe gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.

scholarly journals Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

Nucleotide sequence of the murE gene of Escherichia coli

1989 ◽  
Vol 35 (11) ◽  
pp. 1051-1054 ◽  
Author(s):  
Jing-Song Tao ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Diaminopimelic Acid ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Coding Region ◽  
Base Pairs ◽  
Gene Encoding ◽  
Peptidoglycan Synthesis

The nucleotide sequence of the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli is reported. The coding region consisted of 1413 base pairs and was separated from the ftsI (penicillin-binding protein 3) gene by 61 base pairs. The deduced primary structure of MurE comprised 471 amino acid residues with a molecular mass of 50.6 kilodaltons.Key words: Escherichia coli, murE, peptidoglycan synthesis, diaminopimelic acid adding enzyme.

scholarly journals Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase

1988 ◽  
Vol 249 (2) ◽  
pp. 319-326 ◽  
Author(s):  
I A Anton ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Shikimate Dehydrogenase ◽  
Gene Encoding ◽  
E Coli ◽  
Multifunctional Enzymes ◽  
Aroe Gene ◽  
Terminal Amino

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.

scholarly journals Protection of Mice from Lethal Escherichia coli Infection by Chimeric Human Bactericidal/Permeability-Increasing Protein and Immunoglobulin G1 Fc Gene Delivery

2006 ◽  
Vol 51 (2) ◽  
pp. 724-731 ◽  
Author(s):  
Jindong Chen ◽  
Chengyao Li ◽  
Yuanzhi Guan ◽  
Qingli Kong ◽  
Chen Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Risk ◽  
Gene Delivery ◽  
Bacterial Infections ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
E Coli ◽  
Virus Serotype ◽  
Escherichia Coli Infection ◽  
Risk Patients

ABSTRACT To evaluate the potentiality of applying gene therapy to bacterial infections, especially for preventing infection in high-risk patients, we investigated protection of mice from challenge with lethal Escherichia coli infection by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer of a chimeric BPI23-Fcγ1 gene, which consisted of human bactericidal/permeability-increasing protein (BPI) gene encoding the functional N terminus (amino acid residues 1 to 199) of human BPI and an Fcγ1 gene encoding the Fc segment of human immunoglobulin G1. Here we show that the target protein that was expressed and secreted into the serum of the gene-transferred mice demonstrated the activity of a neutralizing endotoxin, killing E. coli and mediating opsonization. After lethal E. coli infection, the count of bacteria and the levels of endotoxin and proinflammatory cytokines in the gene-transferred mice were decreased. The survival rate of BPI23-Fcγ1 gene-transferred mice markedly increased, especially in conjunction with antibiotics. Our data suggest that AAV2-mediated chimeric BPI23-Fcγ1 gene delivery could potentially be used clinically for the protection and treatment of infection with gram-negative bacteria in high-risk individuals.

scholarly journals Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase

2007 ◽  
Vol 189 (11) ◽  
pp. 3987-3995 ◽  
Author(s):  
Mireille Hervé ◽  
Audrey Boniface ◽  
Stanislav Gobec ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
De Novo ◽  
Diaminopimelic Acid ◽  
Kinetic Properties ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Physiological Properties

ABSTRACT The UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide l-alanyl-γ-d-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide l-Ala-d-Glu and l-Ala very poorly. Replacement of meso-diaminopimelic acid by l-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the ld-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.

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