Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism

Cheng, Wang-Yau; Chen, Yen-Fu; Wang, Hung Jung; Hsu, Kai Cheng; Lin, S.C.; Chen, Tzu Jung; Yang, Jinn-Moon; Wang, Wen-Ching

doi:10.1371/journal.pone.0033481

Cited by 29 publications

(42 citation statements)

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“…A site-directed mutagenesis study on SDH of Escherichia coli showed that it lost substrate-binding activity when the residues were mutated at positions 67, 92, and 107 (T65, J69, and D105, respectively in SDH of H. pylori ) [30]. Our previous study also showed that mutations in the pathway anchor residues (M10, S12, S15, D33, F48, R57, and R132 in SK) reduced the activity of shikimate kinase [14], [31]. These results suggest that the pathway anchors are essential for catalytic reactions and that the mutations on the pathway anchor resides often decrease enzyme activities of SDH and SK (Figs.…”

Section: Resultsmentioning

confidence: 96%

“…Diagrams of the ligands and the pathway anchors for (C) SDH and (D) SK. Residues are colored in red if their mutations lead to loss of enzyme activity [14], [19], [30], [31].…”

Section: Resultsmentioning

confidence: 99%

“…The SK activity was measured by coupling the release of ADP from the SK-catalyzed reaction to the oxidation of NADH using pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) as coupling enzymes [31]. SDH activity was determined by monitoring the formation of NADPH.…”

Section: Methodsmentioning

confidence: 99%

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Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways

et al. 2013

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Many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of “one–disease, one–target, one–drug”. The hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. Furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. Therefore, a new strategy is required for enriching the hit rate and identifying multitarget inhibitors. To address these issues, we propose the pathway-based screening strategy (called PathSiMMap) to derive binding mechanisms for increasing the hit rate and discovering multitarget inhibitors using site-moiety maps. This strategy simultaneously screens multiple target proteins in the same pathway; these proteins bind intermediates with common substructures. These proteins possess similar conserved binding environments (pathway anchors) when the product of one protein is the substrate of the next protein in the pathway despite their low sequence identity and structure similarity. We successfully discovered two multitarget inhibitors with IC50 of <10 µM for shikimate dehydrogenase and shikimate kinase in the shikimate pathway of Helicobacter pylori. Furthermore, we found two selective inhibitors (IC50 of <10 µM) for shikimate dehydrogenase using the specific anchors derived by our method. Our experimental results reveal that this strategy can enhance the hit rates and the pathway anchors are highly conserved and important for biological functions. We believe that our strategy provides a great value for elucidating protein binding mechanisms and discovering multitarget inhibitors.

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Section: Resultsmentioning

confidence: 96%

“…Diagrams of the ligands and the pathway anchors for (C) SDH and (D) SK. Residues are colored in red if their mutations lead to loss of enzyme activity [14], [19], [30], [31].…”

Section: Resultsmentioning

confidence: 99%

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Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways

et al. 2013

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“…Cheng et al also identified the naphthalene-2-sulfonate 25, which had an IC 50 value of 4.9 µM (Fig. 4) [29]. The authors also solved the crystal structure of E114A Hp-SK variant enzyme in complex with 25 (PDB entry 3N2E, 2.53 Å) (Fig.…”

Section: Inhibitors Identified By Screeningmentioning

confidence: 97%

“…Bearing in mind the fact that the elimination mechanism of the DHQ2 enzymes proceeds via enol intermediate 26, which is characterized by ring-flattening between the C2-C3 bond, Frederickson et al [51] designed the first mimetic of the enolate intermediate, 2,3-dehydroquinic acid (29), which proved to be a reversible competitive inhibitor of Mt-DHQ2 and Sc-DHQ2 with K i values of 200 µM and 30 µM, respectively (Fig. 9).…”

Section: Enolate Intermediate Mimeticsmentioning

confidence: 99%

Inhibition of Shikimate Kinase and Type II Dehydroquinase for Antibiotic Discovery: Structure-Based Design and Simulation Studies

González‐Bello

2015

CTMC

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Abstract:The loss of effectiveness of current antibiotics caused by the development of drug resistance has become a severe threat to public health. Current widely used antibiotics are surprisingly targeted at a few bacterial functions -cell wall, DNA, RNA, and protein biosynthesis -and resistance to them is widespread and well identified. There is therefore great interest in the discovery of novel drugs and therapies to tackle antimicrobial resistance, in particular drugs that target other essential processes for bacterial survival. In the past few years a great deal of effort has been focused on the discovery of new inhibitors of the enzymes involved in the biosynthesis of aromatic amino acids, also known as the shikimic acid pathway, in which chorismic acid is synthesized. The latter compound is the synthetic precursor of L-Phe, L-Tyr, L-Phe, and other important aromatic metabolites. These enzymes are recognized as attractive targets for the development of new antibacterial agents because they are essential in important pathogenic bacteria, such as Mycobacterium tuberculosis and Helicobacter pylori, but do not have any counterpart in human cells. This review is focused on two key enzymes of this pathway, shikimate kinase and type II dehydroquinase. An overview of the use of structure-based design and computational studies for the discovery of selective inhibitors of these enzymes will be provided. A detailed view of the structural changes caused by these inhibitors in the catalytic arrangement of these enzymes, which are responsible for the inhibition of their activity, is described.

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Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate

Schoenenberger¹,

Wszołek²,

Meier³

et al. 2018

Biotechnology Journal

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Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by H- and P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.

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Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism

Cited by 29 publications

References 50 publications

Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways

Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways

Inhibition of Shikimate Kinase and Type II Dehydroquinase for Antibiotic Discovery: Structure-Based Design and Simulation Studies

Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate

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