Site-directed mutations and kinetic studies show key residues involved in alkylammonium interactions and reveal two sites for phosphorylcholine in Pseudomonas aeruginosa phosphorylcholine phosphatase

2011 ◽  
Vol 1814 (7) ◽  
pp. 858-863 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lisandro H. Otero ◽  
Cristhian Boetsch ◽  
Carlos E. Domenech ◽  
Fernado D. González-Nilo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinetic Studies ◽  
Phosphorylcholine Phosphatase ◽  
Key Residues

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

2004 ◽  
Vol 71 ◽  
pp. 1-14
Author(s):  
David Leys ◽  
Jaswir Basran ◽  
François Talfournier ◽  
Kamaldeep K. Chohan ◽  
Andrew W. Munro ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Kinetic Studies ◽  
Molecular Assembly ◽  
Conformational Sampling ◽  
Trimethylamine Dehydrogenase ◽  
Key Residues ◽  
Electron Transferring Flavoprotein ◽  
Electron Transfer Complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

scholarly journals Synthesis and In Vitro Screening of Novel Heterocyclic β-d-Gluco- and β-d-Galactoconjugates as Butyrylcholinesterase Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2833
Author(s):  
Krešimir Baumann ◽  
Lorena Kordić ◽  
Marko Močibob ◽  
Goran Šinko ◽  
Srđanka Tomić
Keyword(s):  
Active Site ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
In Vitro Screening ◽  
Sugar Moiety ◽  
Brain Barrier Permeability ◽  
The Central Nervous System ◽  
Key Residues ◽  
Micromolar Range

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood–brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

scholarly journals Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Carlos Eduardo Domenech ◽  
Lisandro Horacio Otero ◽  
Paola Rita Beassoni ◽  
Angela Teresita Lisa
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Adaptive Response ◽  
Metal Ion ◽  
Coordination Geometry ◽  
Choline Metabolism ◽  
Inhibitory Site ◽  
Closed Structure ◽  
Phosphorylcholine Phosphatase ◽  
Hydrolysis Of

Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O)2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure.

Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene

2005 ◽  
Vol 50 (5) ◽  
pp. 251-256 ◽  
Author(s):  
María J. Massimelli ◽  
Paola R. Beassoni ◽  
Marina A. Forrellad ◽  
José L. Barra ◽  
Mónica N. Garrido ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cloning And Expression ◽  
Phosphorylcholine Phosphatase

scholarly journals Characterisation and modelling of a polyhydroxyalkanoate synthase fromAquitaleasp. USM4 reveals a mechanism for polymer elongation

2017 ◽  
Author(s):  
Aik-Hong Teh ◽  
Nyet-Cheng Chiam ◽  
Go Furusawa ◽  
Kumar Sudesh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Class Ii ◽  
Class I ◽  
Common Mechanism ◽  
The Third ◽  
Dimeric Interface ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase ◽  
Key Residues

AbstractPolyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC fromAquitaleasp. USM4, PhaCAq, was characterised and showed aKmof 394 µM and akcatof 476.4 s−1on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC fromCupriavidus necator, PhaCCnrevealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model fromPseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180°. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAqmodel provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

scholarly journals Investigation of the mating system ofPseudomonas aeruginosastrain 1: I. Kinetic studies

1968 ◽  
Vol 12 (1) ◽  
pp. 29-36 ◽  
Author(s):  
J. S. Loutit ◽  
L. E. Pearce ◽  
M. G. Marinus
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mating System ◽  
Genetic Material ◽  
Kinetic Studies ◽  
Potassium Nitrate ◽  
Donor Cells

A method has been developed which will permit the mapping of the genetic material ofPseudomonas aeruginosa. This has been made possible by the use of potassium nitrate in the mating medium and other modifications which have increased the frequency of recombination. In certain circumstances, theilvA12marker may be transferred by more than 1% of the donor cells.

scholarly journals Kinetic studies of allosteric catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa

1998 ◽  
Vol 251 (1-2) ◽  
pp. 528-533 ◽  
Author(s):  
Germaine Sainz ◽  
Catherine Tricot ◽  
Marie-Francoise Foray ◽  
Dominique Marion ◽  
Otto Dideberg ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinetic Studies ◽  
Ornithine Carbamoyltransferase

scholarly journals Class D β-Lactamases: Are They All Carbapenemases?

2014 ◽  
Vol 58 (4) ◽  
pp. 2119-2125 ◽  
Author(s):  
Nuno T. Antunes ◽  
Toni L. Lamoureaux ◽  
Marta Toth ◽  
Nichole K. Stewart ◽  
Hilary Frase ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Clinical Importance ◽  
Kinetic Studies ◽  
Resistance Pattern ◽  
Content Type ◽  
Narrow Spectrum ◽  
Class D ◽  
Life Threatening

ABSTRACTCarbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified inAcinetobacterspp., notably inAcinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur inPseudomonas aeruginosaand are currently classified as narrow-spectrum class D β-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed inEscherichia colistrain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed inA. baumanniiATCC 17978, however, they behave as extended-spectrum β-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum β-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.

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