The AEROPATH project targeting<i>Pseudomonas aeruginosa</i>: crystallographic studies for assessment of potential targets in early-stage drug discovery

Moynié, L.; Schnell, R.; McMahon, S.A.; Sandalova, T.; Boulkerou, Wassila Abdelli; Schmidberger, J.W.; Alphey, M.S.; Cukier, C.D.; Duthie, F.G.; Kopec, J.; Liu, Huanting; Jacewicz, Agata; Hunter, William N.; Naismith, James H.; Schneider, Günter

doi:10.1107/s1744309112044739

Cited by 31 publications

(25 citation statements)

References 71 publications

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“…As part of a multidisciplinary program supporting early stage antimicrobial drug discovery against P. aeruginosa (http://www.aeropath.eu) structural studies of enzymes implicated in the ubiquinone pathway in this organism are carried out [7], [13]. Here, we report the crystal structure of the UbiD-like protein PA0254 of P. aeruginosa in two different crystal forms at 1.95 and 2.3 Å resolution, respectively.…”

Section: Introductionmentioning

confidence: 99%

Structural Insights into the UbiD Protein Family from the Crystal Structure of PA0254 from Pseudomonas aeruginosa

et al. 2013

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The 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) catalyzes the conversion of 3-polyprenyl-4-hydroxybenzoate to 2-polyprenylphenol in the biosynthesis of ubiquinone. Pseudomonas aeruginosa contains two genes (PA0254 and PA5237) that are related in sequence to putative UbiD enzymes. A bioinformatics analysis suggests that the UbiD sequence family can be divided into two subclasses, with PA5237 and PA0254 belonging to different branches of this family. The three-dimensional structure of PA0254 has been determined using single wavelength anomalous diffraction and molecular replacement in two different crystal forms to resolutions of 1.95 and 2.3 Å, respectively. The subunit of PA0254 consists of three domains, an N-terminal α/β domain, a split β-barrel with a similar fold of a family of flavin reductases and a C-terminal α/β domain with a topology characteristic for the UbiD protein family. The middle domain contains a metal binding site adjacent to a large open cleft that may represent the active site. The two protein ligands binding a magnesium ion, His188 and Glu229, invariant in the PA0254 subclass, are also conserved in a corresponding metal site found in one of the FMN binding proteins from the split β-barrel fold family. PA0254 forms, in contrast to the hexameric UbiD from E. coli and P. aeruginosa, a homo-dimer. Insertion of four residues in a loop region in the PA0254 type enzymes results in structural differences that are incompatible with hexamer assembly.

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Section: Introductionmentioning

confidence: 99%

Structural Insights into the UbiD Protein Family from the Crystal Structure of PA0254 from Pseudomonas aeruginosa

et al. 2013

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“…And while structure-based design will indeed contribute to the discovery of new antibacterials -as discussed below and as exemplified by the opportunities implicit in the AEROPATH compilation of Pseudomonas aeruginosa target structures (Moynie et al 2013) -complementary new approaches have emerged to address the central relationship of validating targets with respect to resistance-compromised chemical structure. Proteomic analysis of the resistance response will contribute to an understanding of the overall metabolic adjustment (Lima et al 2013) and the identification of critical protein interaction networks (Zoraghi and Reiner 2013).…”

Section: Will the Discovery Of New Targets Contribute To Overcoming Bmentioning

confidence: 99%

Strategies for Circumventing Bacterial Resistance Mechanisms

Fisher

Johnson

Mobashery

2014

Handbook of Antimicrobial Resistance

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The future practices for the control of bacterial infections are uncertain. The intransigent infection is no longer found just among the immune compromised but is now found both in and out the boundaries of the hospital. Preserving the efficacy of the antibacterials we have, in order to secure the time needed to discover and develop new antibacterials, will require abrupt change: in the way antibacterials are dispensed and disposed, in the criteria used to measure clinical safety and efficacy, in the financial incentives for antibacterial development, and in the understanding of the molecular mechanisms governing the relationship between the antibacterial and the bacterium. This review examines this relationship from the particular perspective of the eventual need to circumvent resistance mechanisms in order to reclaim the lifesaving value of the antibacterial chemical.

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“…EF198106) as described previously (Moynie et al, 2013). The recombinant protein carries a TEV-cleavable His 6 tag at the N-terminus.…”

Section: Cloning Gene Expression and Protein Purificationmentioning

confidence: 99%

“…NADPH binds at the C-terminal end of the -strands, close to helix 8. PA4992 also contains a catalytic triad, Asp66, Tyr71 and Lys94, typical of this enzyme family (Moynie et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the flavoenzyme PA4991 fromPseudomonas aeruginosa

et al. 2016

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The locus PA4991 in Pseudomonas aeruginosa encodes an open reading frame that has been identified as essential for the virulence and/or survival of this pathogenic organism in the infected host. Here, it is shown that this gene encodes a monomeric FAD-binding protein of molecular mass 42.2 kDa. The structure of PA4991 was determined by a combination of molecular replacement using a search model generated with Rosetta and phase improvement by a lowoccupancy heavy-metal derivative. PA4991 belongs to the GR 2 family of FADdependent oxidoreductases, comprising an FAD-binding domain typical of the glutathione reductase family and a second domain dominated by an eightstranded mixed -sheet. Most of the protein-FAD interactions are via the FADbinding domain, but the isoalloxazine ring is located at the domain interface and interacts with residues from both domains. A comparison with the structurally related glycine oxidase and glycerol-3-phosphate dehydrogenase shows that in spite of very low amino-acid sequence identity (<18%) several active-site residues involved in substrate binding in these enzymes are conserved in PA4991. However, enzymatic assays show that PA4991 does not display aminoacid oxidase or glycerol-3-phosphate dehydrogenase activities, suggesting that it requires different substrates for activity.

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The AEROPATH project targetingPseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery

Cited by 31 publications

References 71 publications

Structural Insights into the UbiD Protein Family from the Crystal Structure of PA0254 from Pseudomonas aeruginosa

Structural Insights into the UbiD Protein Family from the Crystal Structure of PA0254 from Pseudomonas aeruginosa

Strategies for Circumventing Bacterial Resistance Mechanisms

Crystal structure of the flavoenzyme PA4991 fromPseudomonas aeruginosa

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