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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/1197
Title: Structural and biochemical analyses reveal that chlorogenic acid inhibits the shikimate pathway
Authors: Neetu N.
Katiki M.
Dev A.
Gaur S.
Tomar, Shailly
Kumar, Pravindra R.Manish
Published in: Journal of Bacteriology
Abstract: Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with KD (equilibrium dissociation constant) values of 6.3 ?M and 0.5 ?M, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 + 21 ?M, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 ?M. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules. IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes. © 2020 American Society for Microbiology. All Rights Reserved.
Citation: Journal of Bacteriology(2020), 202(18): -
URI: https://doi.org/10.1128/JB.00248-20
http://repository.iitr.ac.in/handle/123456789/1197
Issue Date: 2020
Publisher: American Society for Microbiology
Keywords: Chlorogenic acid
Dehydroquinate synthase
Drug discovery
Enzyme structure
Isothermal titration calorimetry
Protein crystallization
Shikimate pathway
Surface plasmon resonance
X-ray crystallography
ISSN: 219193
Author Scopus IDs: 57218693333
57112522900
55620085000
57218693819
57203506001
55064809000
Author Affiliations: Neetu, N., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Katiki, M., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Dev, A., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Gaur, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Tomar, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Kumar, P., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
Funding Details: This work was supported by a grant from the Science and Engineering Research Board (SERB), India (project no. CRG/2018/001022). N. Neetu thanks CSIR; M. Katiki thanks MHRD. We thank the Macromolecular Crystallographic Unit (MCU) facility at IIC, IIT Roorkee, for preliminary data collection and ITC experiments. We also thank the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for data collection. The Synchrotron beamline BM-14 at the European Synchrotron Radiation Facility is sponsored by the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India. N. Neetu, M. Katiki, and P. Kumar designed the research; N. Neetu and M. Katiki performed the research experiments; A. Dev and N. Neetu carried out the cloning; S. Gaur performed the virtual screening; N. Neetu, M. Katiki, and S. Tomar analyzed the SPR data; N. Neetu, M. Katiki, and P. Kumar analyzed data; and N. Neetu, M. Katiki, and P. Kumar wrote the manuscript. All authors reviewed the manuscript. We declare that we have no conflicts of interest with the contents of this article.
Corresponding Author: Kumar, P.; Department of Biotechnology, Indian Institute of Technology RoorkeeIndia; email: pravindra.kumar@bt.iitr.ac.in
Appears in Collections:Journal Publications [BT]

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