scholarly journals In vivocloning of β 1-4 endoglucanase gene ofSerratia liquefaciensusing Muduction and itsin silicoanalysis

2018 ◽  
Author(s):  
S. Gowri Sankar ◽  
J. Asnet Mary ◽  
S. John Vennison ◽  
A. Alwin Prem Anand
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Plant Cell Wall ◽  
Central Axis ◽  
Nucleotide Sequence Analysis ◽  
Cellulolytic Bacterium ◽  
E Coli ◽  
Endoglucanase Gene ◽  
Major Structural Component

AbstractCellulose is the major structural component in the plant cell wall. The bio-degradation of cellulose molecules is facilitated by cellulase. In the present study,in vivocloning of cellulase (β-1, 4 endoglucanase) gene from a cellulolytic bacteriumSerratia liquefaciensintoE. coliDH5α has been performed using mini-Mu phage transduction. The enzyme activity of cloned endoglucanase was 81.2U/mg at optimum temperature (40°C) and 80.2U/mg at optimum pH7, while the wildtype has 65.9U/mg and 64.9U/mg respectively. The conserved domain analysis shows thatS. liquefaciensendoglucanase belongs to GH8 family. The nucleotide sequence analysis of wildtype and cloned endoglucanase shows that mutations were found at residues 51(Lys - Asn), 203(Trp-Cys), 246(Thr-Iso), 260(Gly-Ala) and 288(Phe-Leu). The structural analysis shows the active site of wildtype endoglucanase is a narrow groove which lies parallel to the central axis, whereas cloned endoglucanase is broad and tilted to ∼70° from the central axis. The increased enzyme activity in the cloned endoglucanase is due to the structural modification conferred by changes in amino acid resulting in widening of the cleft in the active site.

An Assay to Predict L-Asparaginase Neutralization and Enzyme Loaded Red Blood Cells to Maintain in Vivo Efficacy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 907-907
Author(s):  
Emmanuelle Dufour ◽  
Christine Saban-Vianey ◽  
Henri Coquelin ◽  
Yann Godfrin
Keyword(s):  
Enzyme Activity ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Neutralizing Antibodies ◽  
Protein A ◽  
The Other ◽  
Total Enzyme Activity ◽  
E Coli ◽  
Asparaginase Activity

Abstract E. coli. L-Asparaginase repeated injections induce immunization. Anti-Asparaginase antibodies can provoke clinical hypersensitivity reactions and/or silently inactivate enzyme activity. Consequently, L-Asparaginase clearance is increased, implying a lack of L-asparagine deamination. Firstly, we developed an assay able to detect the presence of neutralizing factors including anti-Asparaginase antibodies. Next we investigated in a mouse model if loading L-Asparaginase into red blood cells (RBC) may be a way to protect its activity against neutralizing factors. A rabbit was immunized injecting 0.5 mg of L-Asparaginase (167 IU) mixed with Freund’s adjuvant every 3 weeks for 4-fold. The animal was euthanized and the final serum collected. Part of this final serum was immuno-adsorbed onto protein A for IgG antibodies purification. L-Asparaginase activity was measured by monitoring the kinetics of ammonia generation from the hydrolysis of asparagine. This assay was adapted to a biochemistry automated analyzer. When mixed with undiluted serum from the immunized rabbit, L-Asparaginase activity (0.8 to 100 IU/ml) was totally inhibited for all the concentration range within 15 min at 37°C. In the other hand, up to 1/128 serial dilutions of serum totally inhibited 2 IU/ml L-Asparaginase. As a control, undiluted pre-immunization serum from the same animal did not significantly affect L-Asparaginase activity. To identify the neutralizing factors, IgG from serum were purified by protein-A. As performed with serum, successive dilutions of IgG were mixed with 1.25 IU/ml L-Asparaginase. The IgG inhibited enzyme activity at the 1/128 dilution by 97%, thus proving their neutralizing effect on L-Asparaginase. To simulate the presence of neutralizing antibodies in the patient, we injected 7.5 μg of rabbit IgG into OF1 mice. Control mice were injected with phosphate buffered saline (PBS). Twenty minutes later mice either received 80 IU/kg of native E. coli L-Asparaginase or the same dose entrapped into OF1 mouse RBC. L-Asparaginase was loaded into murine RBC by reversible hypotonic dialysis, followed by a resealing step. The RBC thus acts as a bioreactor where plasmatic asparagine enters and is cleaved by the entrapped L-Asparaginase inside the erythrocyte. L-Asparaginase activity inside the erythrocyte was quantified at 68 IU per ml of erythrocytes, and the extracellular enzyme activity was less to 9% of total enzyme activity. Mice were sacrificed 6 hours after the administration of native or encapsulated L-Asparaginase. Free L-Asparaginase was totally inactivated in plasma of anti-Asparaginase IgG pre-treated mice: 0.002 ±0.002 IU/ml vs 0.417 ±0.103 IU/ml in PBS pre-treated mice. In addition, when L-Asparaginase is loaded inside RBC the activity is maintained irrespective of the presence of antibodies (0.798 ±0.126 IU/ml with IgG vs 0.879 ±0.146 IU/ml without). Moreover asparagine was not deaminated in IgG pre-treated mice who received free L-Asparaginase (27 ±1.6 μmol/L), while below 2 μmol/L in all the other groups. In conclusion, this newly developed assay can predict in vivo L-Asparaginase inefficacy. In addition, L-Asparaginase loaded into RBC is protected against neutralizing antibodies and its efficacy is maintained.

scholarly journals In Vivo Acquisition of Ceftriaxone Resistance in Salmonella enterica Serotype Anatum

2003 ◽  
Vol 47 (2) ◽  
pp. 563-567 ◽  
Author(s):  
Lin-Hui Su ◽  
Cheng-Hsun Chiu ◽  
Chishih Chu ◽  
Mei-Hui Wang ◽  
Ju-Hsin Chia ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Salmonella Enterica ◽  
Antimicrobial Agents ◽  
Nucleotide Sequence Analysis ◽  
Molecular Fingerprinting ◽  
Severe Problem ◽  
E Coli ◽  
Tract Infections ◽  
Emergence Of Resistance

ABSTRACT The emergence of resistance to antimicrobial agents within the salmonellas is a worldwide and severe problem. A case of treatment failure due to the emergence of resistance to ceftriaxone in Salmonella enterica serotype Anatum was studied. S. enterica serotype Anatum and Escherichia coli, both of which are susceptible to ceftriaxone, were initially isolated from a diabetic patient hospitalized for the treatment of wound and urinary tract infections. Resistant S. enterica serotype Anatum and E. coli strains were isolated concomitantly 2 weeks after the initiation of ceftriaxone therapy. The patient eventually died of a sepsis caused by the ceftriaxone-resistant salmonella. PCR, nucleotide sequence analysis, and DNA-DNA hybridization identified a bla CTX-M-3 gene located on a 95.1-kb plasmid from the ceftriaxone-resistant isolates of S. enterica serotype Anatum and E. coli. The plasmid was proved to be conjugative. Molecular fingerprinting showed that the susceptible and resistant strains were genetically indistinguishable. The emergence of resistance to ceftriaxone in S. enterica serotype Anatum was due to the in vivo acquisition of a plasmid containing the bla CTX-M-3 gene and was the cause for treatment failure in this patient.

scholarly journals Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lucas A. Gallarato ◽  
Cristhian Boetsch ◽  
Mónica N. Garrido ◽  
Angela T. Lisa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Active Site ◽  
Inorganic Phosphate ◽  
Biochemical Characterization ◽  
E Coli ◽  
Activity Dependent ◽  
Hydrolysis Of ◽  
Molecular Modeling And Docking

Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

scholarly journals In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Sinisa Urban ◽  
Rosanna P. Baker
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Membrane Lipids ◽  
Fold Increase ◽  
Living Cells ◽  
Transmembrane Segment ◽  
Intramembrane Proteases ◽  
Gating Mechanism

Abstract Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.

scholarly journals Identification of a putative flexible loop in Arabidopsis glutathione synthetase

1997 ◽  
Vol 322 (1) ◽  
pp. 241-244 ◽  
Author(s):  
Chang-Lin WANG ◽  
David J. OLIVER
Keyword(s):  
Active Site ◽  
Glutathione Synthetase ◽  
Cadmium Resistance ◽  
Sequence Comparisons ◽  
Vitro Assay ◽  
E Coli ◽  
Flexible Loop ◽  
Conserved Region

Glutathione synthetase catalyses the ATP-dependent ligation of γ-glutamylcysteine with glycine to form glutathione. Amino acid sequence comparisons between the Arabidopsis and the Escherichia coli proteins suggested that a region, identified as a small flexible loop that covers the active site of the E. coli protein, might be conserved in the eukaryotic protein. Three site-directed mutations in the Arabidopsis protein were generated to test this hypothesis. Two mutations within the conserved region (Lys367/Pro368 → Asn/Ser and Gly374 → Val) inactivated the enzyme in an in vivo assay based on cadmium resistance in S. pombe, and in an in vitro assay of the activity of the enzyme expressed in E. coli. A third mutation outside of this conserved region (Leu363 → Glu) had a smaller effect in both assays. These results are consistent with the idea that this glycine-rich loop in the Arabidopsis and E. coli proteins might serve the same function in covering the active site of the enzyme.

scholarly journals The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

1998 ◽  
Vol 180 (7) ◽  
pp. 1947-1950 ◽  
Author(s):  
Renata A. Fabianek ◽  
Hauke Hennecke ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Low Molecular Weight ◽  
Thiol Compounds ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
The Family ◽  
Membrane Bound ◽  
Hydrophilic Domain

ABSTRACT A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels ofBradyrhizobium japonicum holocytochromec 550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.

Construction and targeting of a 5F11-scFv-methioninase fusion protein for antibody-directed enzyme pro-drug therapy (ADEPT) against neuroblastoma

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3043-3043
Author(s):  
J. Hu ◽  
H. Guo ◽  
H. Xu ◽  
N. V. Cheung
Keyword(s):  
Enzyme Activity ◽  
Fusion Protein ◽  
Tumor Therapy ◽  
Immunofluorescent Staining ◽  
Single Chain ◽  
Human Neuroblastoma ◽  
E Coli ◽  
Biodistribution Studies ◽  
Scfv Gene

3043 Background: Recombinant methioninase (METase) depletes methionine and inhibits tumor growth in preclinical models. 5F11 single-chain variable fragment (scFv) can target fusion proteins (e.g. streptavidin (SA), J. Nucl. Med., 45:867, 2004) preferentially to GD2- positive human neuroblastoma (NB). METase targeted to tumors may release methylselenol from the prodrug selenomethionine for tumor therapy. Methods: 5F11-scFv gene fused upstream of METase in pKK-223–3 to form 5F11-scFv-METase. Fusion protein produced in E. coli and purified by chromatography and endotoxin removal retained enzyme activity. Binding of 5F11-scFv-METase to tumor cells was assayed by indirect immunofluorescence and binding kinetics to GD2 analyzed by surface plasmon resonance. Cytotoxicity against human NB cell lines LAN-1, NMB-7 and SK-N-ER was tested using proliferation inhibition and apoptosis assays. Targeting to NB xenografts was studied by biodistribution using 125I-anti-idiotype 1G8 as the secondary tracking antibody. Results: 5F11-scFv-METase was purified to 84% and 94.1% homogeneity by SDS-PAGE and HPLC, respectively. Enzyme activity was 3.6 units/mg protein. Its avidity (KD=1.72×10-7M) as a dimer compared favorably with the monomeric 5F11-scFv (KD=1.07×10- 7M) and tetrameric 5F11-scFv-SA (KD=2.62×10-9M). Immunofluorescent staining of 5F11-scFv-METase was comparable to 5F11-scFv-SA for LAN-1 and NMB-7. In the presence of 20 μM selenomethionine, IC50 of 5F11-scFv-METase was 0.03, 0.02 and 0.02 units/ml for LAN-1, NMB-7 and SK-N-ER, respectively. When treated with 0.3 units/ml of 5F11-scFv-METase and 20 μM selenomethionine, apoptosis was induced in SK-N-ER by 8 hours, peaking at 24 hours. In biodistribution studies, tumor uptake in LAN-1 xenograft averaged 4.07±0.6% injected dose per gram at 48 hours. Conclusions: 5F11-scFv-METase fusion protein retains enzyme activity and immunoreactivity. It targets to tumors in vivo and activates the pro-drug selenomethionine to effect tumor cytotoxicity. Its potential for tumor-selective methionine depletion as well as in ADEPT applications deserves further studies. No significant financial relationships to disclose.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine γ-synthase, E. coli cystathionine β-lyase, and Saccharomyces cerevisiae cystathionine γ-lyase and development of tools for the investigation of their mechanisms and reaction specificity

2009 ◽  
Vol 87 (2) ◽  
pp. 445-457 ◽  
Author(s):  
Ali Farsi ◽  
Pratik H. Lodha ◽  
Jennifer E. Skanes ◽  
Heidi Los ◽  
Navya Kalidindi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Affinity Purification ◽  
Catalytic Efficiency ◽  
Active Site Residues ◽  
E Coli ◽  
Transsulfuration Pathway ◽  
Reaction Specificity

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine γ-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share ∼30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/Kml-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/Kml-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The ΔmetB and ΔmetC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

Factors influencing the in vivo stability of L-serine deaminase activity in E. coli K12

1978 ◽  
Vol 24 (12) ◽  
pp. 1607-1613
Author(s):  
R. D. Beeraj ◽  
J. F. Morris ◽  
E. B. Newman
Keyword(s):  
Enzyme Activity ◽  
Nitrogen Source ◽  
Inorganic Nitrogen ◽  
Intact Cells ◽  
Inorganic Nitrogen Source ◽  
E Coli ◽  
Organic Nitrogen Source ◽  
Serine Deaminase ◽  
In Cells

L-Serine deaminase (L-SD) is unstable in intact cells of Escherichia coli K12. The extent of this instability is dependent on the nitrogen content of the medium in which the enzyme is synthesized, and on that in which it is tested. Enzyme activity in cells grown with an inorganic nitrogen source is unstable in the presence of inorganic nitrogen; enzyme activity in cells grown with an organic nitrogen source is unstable in the presence of the amino acids glycine and leucine.

scholarly journals Structural analysis of E. coli 6-carboxytetrahydropterin synthase

2014 ◽  
Vol 70 (a1) ◽  
pp. C462-C462
Author(s):  
Asaithambi Killivalavan ◽  
Kyung Seo ◽  
Ningning Zhuang ◽  
Young Park ◽  
Kon Lee
Keyword(s):  
Active Site ◽  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Wild Type ◽  
Specific Enzyme ◽  
Aromatic Residues ◽  
E Coli

The Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homolog of 6-pyruvoyl tetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro rather than the genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. We have determined crystal structures of a wild type apo-eCTPS and a Cys27Ala mutant eCTPS complexed with sepiapterin up to 2.3 and 2.5 Å, respectively. The structures are highly conserved at the active site and the Zn2+ binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologs revealed that two specific residues Trp51 and Phe55, not existing in the mammalian PTPS, kept the substrate bound by stacking it with their side chains. Replacements of these two residues by site-directed mutagenesis to the residues, Met and Leu, existing only in mammalian PTPS, converted the eCTPS to have the mammalian PTPS activity. Our studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and for the specific enzyme activity different from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologs.

Sign in / Sign up

Export Citation Format

Share Document