scholarly journals A Selective, Dual Emission β-Alanine Aminopeptidase Activated Fluorescent Probe for the Detection of Pseudomonas aeruginosa, Burkholderia cepacia, and Serratia marcescens

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3550 ◽  
Author(s):  
Linda Váradi ◽  
Elias Y. Najib ◽  
David E. Hibbs ◽  
John D. Perry ◽  
Paul W. Groundwater
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Serratia Marcescens ◽  
Burkholderia Cepacia ◽  
Peptide Bond ◽  
Dual Emission ◽  
Enzyme Substrate ◽  
Alanine Aminopeptidase ◽  
Alanyl Aminopeptidase ◽  
Hydrolysis Of ◽  
Flavone Derivative

Selective detection of β-alanyl aminopeptidase (BAP)-producing Pseudomonas aeruginosa, Serratia marcescens, and Burkholderia cepacia was achieved by employing the blue-to-yellow fluorescent transition of a BAP-specific enzyme substrate, 3-hydroxy-2-(p-dimethylaminophenyl)flavone derivative, incorporating a self-immolative linker to β-alanine. Upon cellular uptake and accumulation of the substrate by viable bacterial colonies, blue fluorescence was generated, while hydrolysis of the N-terminal peptide bond by BAP resulted in the elimination of the self-immolative linker and the restoration of the original fluorescence of the flavone derivative.

scholarly journals Extracellular proteolytic activation of Pseudomonas aeruginosa aminopeptidase (PaAP) and insight into the role of its non-catalytic N-terminal domain

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252970
Author(s):  
Itschak Axelrad ◽  
Mary Safrin ◽  
Rivka Cahan ◽  
Sang-Jin Suh ◽  
Dennis E. Ohman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Domain ◽  
Peptide Bond ◽  
Proteolytic Activation ◽  
Terminal Domain ◽  
Natural Peptides ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Shed Light

Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.

scholarly journals GES-2, a Class A β-Lactamase fromPseudomonas aeruginosa with Increased Hydrolysis of Imipenem

2001 ◽  
Vol 45 (9) ◽  
pp. 2598-2603 ◽  
Author(s):  
Laurent Poirel ◽  
Gerhard F. Weldhagen ◽  
Thierry Naas ◽  
Christophe De Champs ◽  
Michael G. Dove ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Blood Cultures ◽  
Class A ◽  
Omega Loop ◽  
Cephalosporin Resistance ◽  
Lactamase Gene ◽  
Hydrolysis Of

ABSTRACT Pseudomonas aeruginosa GW-1 was isolated in 2000 in South Africa from blood cultures of a 38-year-old female who developed nosocomial pneumonia. This isolate harbored a self-transferable ca. 100-kb plasmid that conferred an expanded-spectrum cephalosporin resistance profile associated with an intermediate susceptibility to imipenem. A β-lactamase gene, bla GES-2, was cloned from whole-cell DNA of P. aeruginosa GW-1 and expressed in Escherichia coli. GES-2, with a pI value of 5.8, hydrolyzed expanded-spectrum cephalosporins, and its substrate profile was extended to include imipenem compared to that of GES-1, identified previously in Klebsiella pneumoniae. GES-2 activity was less inhibited by clavulanic acid, tazobactam and imipenem than GES-1. The GES-2 amino acid sequence differs from that of GES-1 by a glycine-to-asparagine substitution in position 170 located in the omega loop of Ambler class A enzymes. This amino acid change may explain the extension of the substrate profile of the plasmid-encoded β-lactamase GES-2.

Enhancing enzymatic hydrolysis of coconut husk through Pseudomonas aeruginosa AP 029/GLVIIA rhamnolipid preparation

2017 ◽  
Vol 237 ◽  
pp. 20-26 ◽  
Author(s):  
Cynthia Kérzia Costa de Araújo ◽  
Alan de Oliveira Campos ◽  
Carlos Eduardo de Araújo Padilha ◽  
Francisco Canindé de Sousa Júnior ◽  
Ruthinéia Jéssica Alves do Nascimento ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzymatic Hydrolysis ◽  
Coconut Husk ◽  
Hydrolysis Of

scholarly journals Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

2007 ◽  
Vol 407 (2) ◽  
pp. 207-217 ◽  
Author(s):  
Claudia Massa ◽  
Mads H. Clausen ◽  
Jure Stojan ◽  
Doriano Lamba ◽  
Cristiana Campa
Keyword(s):  
Mode Of Action ◽  
Burkholderia Cepacia ◽  
Time Course ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Mechanism ◽  
Processive Enzyme ◽  
Chain Lengths ◽  
Methylation Patterns ◽  
Hydrolysis Of ◽  
Hydrolase Family

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

THE POTENTIAL OF BACTERIA FOR THE MICROBIAL CONTROL OF GRASSHOPPERS AND LOCUSTS

1997 ◽  
Vol 129 (S171) ◽  
pp. 147-156 ◽  
Author(s):  
B. Zelazny ◽  
M.S. Goettel ◽  
B. Keller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacillus Thuringiensis ◽  
Serratia Marcescens ◽  
Schistocerca Gregaria ◽  
Locusta Migratoria ◽  
Microbial Control ◽  
Control Agent ◽  
Promising Agent ◽  
Laboratory Populations ◽  
The Many

AbstractBacteria have been implicated in disease epizootics observed in field populations and laboratory-reared locusts and grasshoppers. Two species [Serratia marcescens Bizio and Pseudomonas aeruginosa (Schroeter) Migula] consistently infect locusts when ingested with food and can spread in laboratory populations. However, research on developing these organisms for microbial control of locusts and grasshoppers begun in the 1950s has not been continued. In recent years strains of Bacillus thuringiensis Berliner have been studied for activity against locusts and grasshoppers. Results of additional trials by the authors are reported. Among 393 B. thuringiensis isolates and 93 preparations of other sporeforming bacteria fed to nymphs of Locusta migratoria (L.) and/or Schistocerca gregaria Forsk., none has shown any pathogenicity to the insects. The recent discovery of novel B. thuringiensis strains active against various diverse pests and the many properties of a sporeforming bacterium that satisfy the requirements for a microbial control agent, and the development of Serratia entomophila as a promising agent for control of grass grubs, provide incentive to continue the search for an orthopteran-active sporeforming bacterium and to re-investigate the potential of non-sporeforming bacterial pathogens as microbial control agents of grasshoppers and locusts.

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