Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

Meneely, Kathleen M.; Luo, Qianyi; Dhar, Prajnaparamita; Lamb, Audrey L.

doi:10.1016/j.abb.2013.07.026

Cited by 14 publications

(31 citation statements)

References 36 publications

(85 reference statements)

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“…The pchA gene was cloned as previously described (18) . The PchA variants were produced using QuikChange (Agilent) with wildtype pchA plasmid as the template and the following primers: D310E (5′-GAC AGC GCC AAG GAG AGG CAC GAA CAC-3′) and A375T (5′-GCA TCC GAC CCC CAC GGT GGG CGG CTA CC-3′) and complementary reverse primers for each variant.…”

Section: Methodsmentioning

confidence: 99%

“…Wildtype and variant PchA was purified as previously described (18) with a yield of 16–74 mg/L of culture for the variants. The P. aeruginosa isochorismate-pyruvate lyase PchB used in the coupled assay with PchA to convert isochorismate to salicylate was purified as previously described (23) .…”

Section: Methodsmentioning

confidence: 99%

“…Ferrer and colleagues (17) have suggested that the general base is a glutamic acid (E313 in PchA), but more recently Meneely et. al report a model based on pH/pD rate profiles and mutagenesis that is most consistent with the lysine general base and the glutamic acid general acid (18) .…”

Section: Introductionmentioning

confidence: 99%

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Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Meneely

Luo

Lamb

2013

Archives of Biochemistry and Biophysics

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The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Meneely

Luo

Lamb

2013

Archives of Biochemistry and Biophysics

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“…Chorismate was isolated from Klebisella pneumonia 62-1 [65] with only minor changes, as previously described [14]. …”

Section: Methodsmentioning

confidence: 99%

Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism

Meneely

Luo

Riley

et al. 2014

Bioorganic & Medicinal Chemistry

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Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismatepyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin. Compounds identified in the screen are high nanomolar to low micromolar inhibitors of the enzyme and produce growth inhibition in PAO1 P. aeruginosa in the millimolar range under iron-limiting conditions. The identified compounds were also tested for enzymatic inhibition of E. coli chorismate mutase, a protein of similar fold and similar chemistry, and of Y. enterocolitica salicylate synthase, a protein of differing fold but catalyzing the same lyase reaction. In both cases, subsets of the inhibitors from the screen were found to be inhibitory to enzymatic activity (mutase or synthase) in the micromolar range and capable of growth inhibition in their respective organisms (E. coli or Y. enterocolitica).

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“…In the second reaction, which occurs in the same active site, pyruvate is eliminated from isochorismate via an intramolecular [3,3]-sigmatropic rearrangement to afford salicylic acid via bicyclic transition state TS-2 . 6a, 10, 11,12 …”

Section: Introductionmentioning

confidence: 99%

Synthesis of Transition-State Inhibitors of Chorismate Utilizing Enzymes from Bromobenzene cis-1,2-Dihydrodiol

et al. 2017

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In order to survive in a mammalian host, Mycobacterium tuberculosis (Mtb) produces aryl-capped siderophores known as the mycobactins for iron acquisition. Salicylic acid is a key building block of the mycobactin core and is synthesized by the bifunctional enzyme MbtI, which converts chorismate into isochorismate via a SN2″ reaction followed by further transformation into salicylate through a [3,3]-sigmatropic rearrangement. MbtI belongs to a family of chorismate-utilizing enzymes (CUEs) that have conserved topology and active site residues. The transition-state inhibitor 1 described by Bartlett, Kozlowski and co-workers is the most potent reported inhibitor to date of CUEs. Herein we disclose a concise asymmetric synthesis and the accompanying biochemical characterization of 1 along with three closely related analogues beginning from bromobenzene cis-1S,2S-dihydrodiol produced through microbial oxidation that features a series of regio- and stereoselective transformations for introduction of the C-4 hydroxy and C-6 amino substituents. The flexible synthesis enables late–stage introduction of the carboxy group and other bioisosteres at the C-1 position as well as installation of the enol-pyruvate side chain at the C-5 position.

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Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

Cited by 14 publications

References 36 publications

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism

Synthesis of Transition-State Inhibitors of Chorismate Utilizing Enzymes from Bromobenzene cis-1,2-Dihydrodiol

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