Synthesis of Bisubstrate Inhibitors of Porphobilinogen Synthase from <i>Pseudomonas aeruginosa</i>

Gacond, Sabine; Frere, F.; Nentwich, Merle; Faurite, Jean-Philippe; Frankenberg‐Dinkel, Nicole; Neier, Reinhard

doi:10.1002/cbdv.200790024

Cited by 9 publications

(4 citation statements)

References 28 publications

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“…PBGS activity was quantified by a modified Ehrlich's test, based on the reaction between the product PBG with 4-(dimethylamino)-benzaldehyde (10,15). The Michaelis-Menten constant (K m ), the maximal velocity (V max ), and the catalytic constant (k cat ) were determined by measuring the constant rate of PBG formation for 0 to 10 mM ALA and iteratively optimized Lineweaver-Burk plots by using the SigmaPlot (version 8.0) and Enzyme Kinetics (version 1.1) programs.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Heme Biosynthetic Pseudomonas aeruginosa Porphobilinogen Synthase in Complex with the Antibiotic Alaremycin

Heinemann

Schulz

Schubert

et al. 2010

Antimicrob Agents Chemother

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The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a K i of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-Å resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed.

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Section: Methodsmentioning

confidence: 99%

Structure of the Heme Biosynthetic Pseudomonas aeruginosa Porphobilinogen Synthase in Complex with the Antibiotic Alaremycin

Heinemann

Schulz

Schubert

et al. 2010

Antimicrob Agents Chemother

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“…Although we and others have investigated active site directed inhibition of PBGS, potent and species specific active site inhibitors of PBGS from a human pathogen have not been reported [19,[25][26][27][28][29][30][31]. Species-selective inhibitors that function by stabilizing the hexamer of pea PBGS and compounds that stabilize the hexamer of Y. enterocolitica PBGS were discovered by computational docking of small molecule libraries from Life Chemicals, Inc., to the hexamer-specific surface cavity using the program GLIDE and other components of the Schrodinger software suite [32,33].…”

Section: Allosteric Inhibition Of Pbgs As a Foundation For Antimicrobmentioning

confidence: 99%

Morpheeins – A New Pathway For Allosteric Drug Discovery

Jaffe¹

2010

TOPROCJ

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Abstract:The morpheein model of allosteric regulation can be applied as a novel approach to the discovery of small molecule allosteric modulators of protein function. Morpheeins are homo-oligomeric proteins where, under physiological conditions, the oligomer can dissociate, the dissociated units can change conformation, and the altered conformational state can reassociate to a structurally and functionally distinct oligomer. This phenomenon serves as a basis for allostery, as a basis for conformational diseases, as a basis for drug discovery, and may be applicable to personalized medicine such as in the prediction of drug side effects. Each of these relationships has been established for the prototype morpheein, porphobilinogen synthase, where the conformational disease is a porphyria and the drug application is in antimicrobial discovery. These data are presented along with a discussion of other drug targets for which the morpheein model of allostery may apply. Such targets include HIV integrase, TNF , -tryptase, and p53.

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“…Unfortunately, it was found to be a weak inhibitor of dAdo kinase I, with a calculated K iapp of 28 M against ATP at a fixed dAdo concentration of 0.01 mM. In contrast, dAp 5 A, with one more phosphate group, appeared to be a potent and specific inhibitor for dAdo kinases, with a K iapp of 2.7 M for dAdo kinase I 78 .…”

Section: Deoxynucleoside Kinasesmentioning

confidence: 93%

“…In an aim to increase the knowledge of the active site of the enzyme and to elucidate its mechanism, Neier et al developed bisubstrate analogs (Scheme 1) which incorporated two -keto carboxylic groups for recognition with the enzyme's active site, and were attached together by several linkers 5 . These bisubstrates were evaluated as potential inhibitors of Mg 2+ -dependent PBGS from Pseudomonas aeruginosa (ALA K m = 0.33 mM).…”

Section: Porphobilinogen Synthasementioning

confidence: 99%