scholarly journals Insights into Enzymic Catalysis from Studies on Dihydrofolate Reductases

Pteridines ◽  
1989 ◽  
Vol 1 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Stephen J. Benkovic ◽  
Joseph A. Adams ◽  
Carol A. Fierke ◽  
Adel M. Naylor
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Structural Similarity ◽  
E Coli ◽  
Energy Profiles ◽  
Dihydrofolate Reductases ◽  
Cellular Dna ◽  
High Degree ◽  
Conserved Amino Acids

Summary The role of DHFR in the maintenance of cellular DNA has sparked wide interest in the structure and dynamics of this enzyme. Kinetic studies of specific amino acid replacements on the enzyme isolated from E. coli has proved useful in the detailing of hydrophobic and ionic interactions both proximal and distal to the site of chemical transformation (e. g. Phe-31, Leu-54 and Arg-44). Despite the low sequence homology shared by the E. coli and L. easei enzymes, the free energy profiles are surprisingly comparable. This probably is the result of the high degree of structural similarity of the active site surfaces, but the deleterious effects of subtle replacements (e. g. Leu-54-Ile) at strictly conserved amino acids underscore the latters unique role in attaining the required catalytic efficiency for the enzyme.

Control of disinfection and halogenated disinfection byproducts by the electrochemical process

2007 ◽  
Vol 55 (12) ◽  
pp. 213-219 ◽  
Author(s):  
Y.J. Jung ◽  
B.S. Oh ◽  
J.W. Kang ◽  
M.A. Page ◽  
M.J. Phillips ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Disinfection Byproducts ◽  
Electrochemical Process ◽  
Free Chlorine ◽  
Haloacetic Acids ◽  
Oh Radicals ◽  
Inactivation Kinetic ◽  
E Coli ◽  
Electrochemical Processes

The aim of this study was to investigate some aspects of the performance of electrochemical process as an alternative disinfection strategy, while minimising DBPs, for water purification. The study of electrochemical processes has shown free chlorine to be produced, but smaller amounts of stronger oxidants, such as ozone, hydrogen peroxide and OH radicals (•OH), were also generated. The formation of mixed oxidants increased with increasing electric conductivity, but was limited at conductivities greater than 0.6 mS/cm. Using several microorganisms, such as E. coli and MS2 bacteriophage, inactivation kinetic studies were performed. With the exception of free chlorine, the role of mixed oxidants, especially OH radicals, was investigated for enhancement of the inactivation rate. Additionally, the formation and reduction of DBPs was studied by monitoring the concentration of haloacetic acids (HAAs) during the process.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Polyaspartamide Functionalized Catechol-Based Hydrogels Embedded with Silver Nanoparticles for Antimicrobial Properties

Polymers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1188 ◽  
Author(s):  
Milène Tan ◽  
Youngjin Choi ◽  
Jaeyun Kim ◽  
Ji-Heung Kim ◽  
Katharina Fromm
Keyword(s):  
Antimicrobial Properties ◽  
Kinetic Studies ◽  
Bactericidal Effect ◽  
Release Kinetic ◽  
E Coli ◽  
Dispersed Particles ◽  
Time Period ◽  
Long Time ◽  
20 Nm

In this study, polyaspartamide-based hydrogels were synthesized by boron-catechol coordination followed by incorporation of AgNPs into the materials. Free catechol moieties were exploited to produce AgNPs. TEM analyses displayed AgNPs of less than 20 nm in diameter and with minimum aggregation, attesting the role of hydrogels to act as an efficient template for the production of dispersed particles. XRD analyses confirmed the mean particle size using the Scherrer equation. Release kinetic studies were performed in DMEM medium, showing a slow release over a long time-period. Finally, the MIC and MBC were determined, demonstrating a bacteriostatic and bactericidal effect against Gram-positive S. aureus and Gram-negative E. coli.

scholarly journals Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Susann Skagseth ◽  
Tony Christopeit ◽  
Sundus Akhter ◽  
Annette Bayer ◽  
Ørjan Samuelsen ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Global Public Health ◽  
Equilibrium Dissociation Constant ◽  
Inhibitor Binding ◽  
Class A ◽  
Class D ◽  
Equilibrium Dissociation ◽  
Structural Insights

ABSTRACT Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 μM; KD = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

2010 ◽  
Vol 54 (11) ◽  
pp. 4556-4560 ◽  
Author(s):  
Hedi Mammeri ◽  
Hélène Guillon ◽  
François Eb ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Analysis ◽  
Carbapenem Resistance ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
E Coli ◽  
Genes Encoding ◽  
High Level ◽  
Biochemical Comparison ◽  
Outer Membrane Permeability

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

scholarly journals Protein motions during catalysis by dihydrofolate reductases

2006 ◽  
Vol 361 (1472) ◽  
pp. 1317-1321 ◽  
Author(s):  
Rudolf K Allemann ◽  
Rhiannon M Evans ◽  
Lai-hock Tey ◽  
Giovanni Maglia ◽  
Jiayun Pang ◽  
...  
Keyword(s):  
Hydrogen Transfer ◽  
Isotope Effects ◽  
Kinetic Studies ◽  
Evolutionary Pattern ◽  
E Coli ◽  
Protein Motions ◽  
Steady State Kinetic ◽  
Dihydrofolate Reductases ◽  
Hydrogen Deuterium ◽  
Reduced Nicotinamide Adenine Dinucleotide

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima . We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

scholarly journals Insertion sequence-related genetic variation in resting Escherichia coli K-12.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 721-730 ◽  
Author(s):  
T Naas ◽  
M Blot ◽  
W M Fitch ◽  
W Arber
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Insertion Sequence ◽  
Ancestral Population ◽  
E Coli ◽  
Rich Media ◽  
Reduced Rate ◽  
K 12 ◽  
High Degree

Abstract Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.

scholarly journals Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in Chryseobacterium meningosepticum

2000 ◽  
Vol 44 (7) ◽  
pp. 1878-1886 ◽  
Author(s):  
Samuel Bellais ◽  
Daniel Aubert ◽  
Thierry Naas ◽  
Patrice Nordmann
Keyword(s):  
Amino Acid ◽  
Broad Spectrum ◽  
Bacterial Species ◽  
Catalytic Efficiency ◽  
Amino Acid Identity ◽  
Relative Molecular Mass ◽  
Acid Identity ◽  
E Coli ◽  
Class B ◽  
Conserved Amino Acids

ABSTRACT Although the carbapenem-hydrolyzing β-lactamase (CHβL) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHβL gene, bla GOB-1, was cloned from another C. meningosepticum clinical isolate (PINT). The G+C content of bla GOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression inEscherichia coli DH10B yields a mature CHβL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHβLs, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHβLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like β-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticumisolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like β-lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHβLs that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.

scholarly journals The Large Resolvase TndX Is Required and Sufficient for Integration and Excision of Derivatives of the Novel Conjugative Transposon Tn5397

2000 ◽  
Vol 182 (23) ◽  
pp. 6577-6583 ◽  
Author(s):  
Hongmei Wang ◽  
Peter Mullany
Keyword(s):  
Hot Spots ◽  
Activity Analysis ◽  
The Novel ◽  
Conjugation System ◽  
Conjugative Transposon ◽  
Site Specific ◽  
E Coli ◽  
Derivatives Of ◽  
Conserved Amino Acids

ABSTRACT Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and fromBacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role oftndX, the gene was cloned and the protein was expressed inEscherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion inE. coli.

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