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Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis

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Abstract

Chorismate serves as a crucial precursor for the synthesis of many aromatic compounds essential for the survival and virulence in various bacteria and protozoans. Chorismate synthase, a vital enzyme in the shikimate pathway, is responsible for the formation of chorismate from enolpyruvylshikimate-3-phosphate (EPSP). Moraxella catarrhalis is reported to be resistant to many beta-lactam antibiotics and causes chronic ailments such as otitis media, sinusitis, laryngitis, and bronchopulmonary infections. Here, we have cloned the aroC gene from Moraxella catarrhalis in pET28c and heterologously produced the chorismate synthase (~ 43 kDa) in Escherichia coli BL21(DE3) cells. We have predicted the three-dimensional structure of this enzyme and used the refined model for ligand-based virtual screening against Supernatural Database using PyRx tool that led to the identification of the top three molecules (caffeic acid, gallic acid, and o-coumaric acid). The resultant protein–ligand complex structures were subjected to 50 ns molecular dynamics (MD) simulation using GROMACS. Further, the binding energy was calculated by MM/PBSA approach using the trajectory obtained from MD simulation. The binding affinities of these compounds were validated with ITC experiments, which suggest that gallic acid has the highest binding affinity amongst these three phytochemicals. Together, these results pave the way for the use of these phytochemicals as potential anti-bacterial compounds.

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Acknowledgments

NN thanks to CSIR, MS, thanks to MHRD; JKM, thanks to DBT for providing financial assistance. The authors also thank the Macromolecular Crystallographic Facility (MCU) at IIC, Indian Institute of Technology Roorkee, for using the facility to carry out experiments.

Funding

This work was supported by a grant from the Science and Engineering Research Board (SERB), India (Project No. CRG/2018/001022).

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  1. Neetu Neetu and Monica Sharma have contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India

    Neetu Neetu, Monica Sharma, Jai Krishna Mahto & Pravindra Kumar

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  1. Neetu Neetu
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  2. Monica Sharma
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Contributions

NN, MS, and PK designed the study. NN, MS, JKM performed the cloning, expression, purification, and biophysical experiments. MS, JKM performed in-silico studies and analysis. NN, MS, JKM, and PK wrote the manuscript. All the authors have approved the final version of the manuscript.

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Correspondence to Pravindra Kumar.

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Neetu, N., Sharma, M., Mahto, J.K. et al. Biophysical and In-Silico Studies of Phytochemicals Targeting Chorismate Synthase from Drug-Resistant Moraxella Catarrhalis. Protein J 39, 449–460 (2020). https://doi.org/10.1007/s10930-020-09923-y

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