scholarly journals Mutant TEM β-Lactamase Producing Resistance to Ceftazidime, Ampicillins, and β-Lactamase Inhibitors

2002 ◽  
Vol 46 (3) ◽  
pp. 646-653 ◽  
Author(s):  
Sergei Vakulenko ◽  
Dasantila Golemi
Keyword(s):  
Amino Acid ◽  
Bacterial Resistance ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Dna Shuffling ◽  
Amino Acid Substitutions ◽  
Triple Mutation ◽  
Low Levels ◽  
Clinically Significant ◽  
Additional Amino Acid

ABSTRACT A derivative of the TEM-1 β-lactamase producing clinically significant levels of resistance to ceftazidime and β-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection. This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to β-lactamase inhibitors. Although the Glu104Lys substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime. Kinetic studies of the purified β-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (k cat/Km ) of ceftazidime. The increases in the Ki values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibility of β-lactam resistance with these inhibitors.

The modulation of HBsAg level by sI126T is affected by additional amino acid substitutions in the S region of HBV

2019 ◽  
Vol 75 ◽  
pp. 104006
Author(s):  
Lingyuan He ◽  
Mingze Su ◽  
Guomin Ou ◽  
Luwei Wang ◽  
Juan Deng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Hbsag Level ◽  
Additional Amino Acid

scholarly journals Chlorella Virus-Encoded Deoxyuridine Triphosphatases Exhibit Different Temperature Optima

2005 ◽  
Vol 79 (15) ◽  
pp. 9945-9953 ◽  
Author(s):  
Yuanzheng Zhang ◽  
Hideaki Moriyama ◽  
Kohei Homma ◽  
James L. Van Etten
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Divalent Cations ◽  
Catalytic Efficiency ◽  
Amino Acid Substitutions ◽  
Temperature Optimum ◽  
Docking Simulations ◽  
Chlorella Virus ◽  
Temperature Optima

ABSTRACT A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg2+ for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a Km of 11.7 μM, a turnover k cat of 6.8 s−1, and a catalytic efficiency of k cat/Km = 5.8 × 105 M−1 s−1. dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37°C) than PBCV-1 dUTPase (50°C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81→Ser81 and Thr84→Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84→Arg84, Glu81→Ser81, and Glu81→Ser81 plus Thr84→Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55°C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

scholarly journals Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum β-Lactamase CTX-M-9

2006 ◽  
Vol 50 (2) ◽  
pp. 731-738 ◽  
Author(s):  
J. Delmas ◽  
F. Robin ◽  
F. Carvalho ◽  
C. Mongaret ◽  
R. Bonnet
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Random Mutagenesis ◽  
Evolutionary Process ◽  
Catalytic Efficiency ◽  
Kinetic Constants ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Evolutionary Potential

ABSTRACT A random mutagenesis technique was used to predict the evolutionary potential of β-lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1- to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k cat value than CTX-M-9 against ceftazidime. The other two had 75- to 300-fold-lower Km values than CTX-M-9 against ceftazidime. The current emergence of CTX-M β-lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.

scholarly journals Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease

2021 ◽  
Author(s):  
Elizabeth A. MacDonald ◽  
Gary Frey ◽  
Mark N. Namchuk ◽  
Stephen C. Harrison ◽  
Stephen M. Hinshaw ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antiviral Drug ◽  
Catalytic Efficiency ◽  
Peptide Sequence ◽  
Structural Proteins ◽  
Amino Acid Substitutions ◽  
Cleavage Sites ◽  
Main Protease ◽  
Scissile Bond ◽  
Viral Polyprotein

ABSTRACTThe main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. It is required for infection, it cleaves the viral polyprotein at multiple sites, and it is conserved among coronaviruses and distinct from human proteases. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes substrates and how the peptide sequence can dictate catalytic efficiency by influencing the position of the scissile bond. One peptide, constituting the junction between viral non-structural proteins 8 and 9 (nsp8/9), has P1’ and P2’ residues that are unique among SARS-CoV-2 cleavage sites but conserved among nsp8/9 junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1’ or P2’ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

scholarly journals Mutation Spectra and the Neutrality of Mutations

1974 ◽  
Vol 27 (3) ◽  
pp. 309 ◽  
Author(s):  
J Langridge
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Amino Acid Replacement ◽  
Enzyme Function ◽  
Substrate Affinity ◽  
Amino Acid Substitutions ◽  
Galactosidase Activity ◽  
Immunological Tests ◽  
Normal Enzyme ◽  
Mutation Spectra

The effect of amino acid replacements on enzyme function was studied in the tJ-galactosidase of Escherichia coli. Mutants possessing 50% or less of normal enzyme activity were isolated and examined. Of 733 amino acid substitutions calculated to have occurred, only 11 reduced tJ-galactosidase activity below 50 %. These mutations were expressed because they greatly impaired the substrate affinity or catalytic efficiency of the enzyme. The inertness of the enzyme to amino acid replacement was confirmed by immunological tests of tJ-galactosidase molecules changed in amino acid sequence by suppression.

scholarly journals E240V Substitution Increases Catalytic Efficiency toward Ceftazidime in a New Natural TEM-Type Extended-Spectrum β-Lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens Clinical Isolates

2007 ◽  
Vol 52 (3) ◽  
pp. 915-919 ◽  
Author(s):  
Mariagrazia Perilli ◽  
Giuseppe Celenza ◽  
Francesca De Santis ◽  
Cristina Pellegrini ◽  
Chiara Forcella ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Serratia Marcescens ◽  
Southern Italy ◽  
Enterobacter Aerogenes ◽  
Catalytic Efficiency ◽  
Amino Acid Substitutions ◽  
Threonine Residue ◽  
Extended Spectrum ◽  
The Stability

ABSTRACT The aim of this study was to characterize a novel extended-spectrum β-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149T182M, in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The bla TEM-149 and bla TEM-149/T182M genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all β-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149T182M enzymes, a reduction of the catalytic efficiency for the TEM-149T182M mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 β-lactamase.

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