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Structural and Biochemical Studies on Klebsiella Pneumoniae Enoyl-ACP Reductase (FabI) Suggest Flexible Substrate Binding Site

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Abstract

Klebsiella pneumoniae, a bacterial pathogen infamous for antibiotic resistance, is included in the priority list of pathogens by various public health organizations due to its extraordinary ability to develop multidrug resistance. Bacterial fatty acid biosynthesis pathway-II (FAS-II) has been considered a therapeutic drug target for antibacterial drug discovery. Inhibition of FAS-II enzyme, enoyl-acyl carrier protein reductase, FabI, not only inhibits bacterial infections but also reverses antibiotic resistance. Here, we characterized Klebsiella pneumoniae FabI (KpFabI) using complementary experimental approaches including, biochemical, x-ray crystallography, and molecular dynamics simulation studies. Biophysical studies shows that KpFabI organizes as a tetramer molecular assembly in solution as well as in the crystal structure. Enzyme kinetics studies reveal a distinct catalytic property towards crotonyl CoA and reducing cofactor NADH. Michaelis-Menten constant (Km) values of substrates show that KpFabI has higher preference towards NADH as compared to crotonyl CoA. The crystal structure of tetrameric apo KpFabI folds into a classic Rossman fold in which β-strands are sandwiched between α-helices. A highly flexible substrate binding region is located toward the interior of the tetrameric assembly. Thermal stability assay on KpFabI with its substrate shows that the flexibility is primarily stabilized by cofactor NADH. Moreover, the molecular dynamics further supports that KpFabI has highly flexible regions at the substrate binding site. Together, these findings provide evidence for highly dynamic substrate binding sites in KpFabI, therefore, this information will be vital for specific inhibitors discovery targeting Klebsiella pneumoniae.

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Acknowledgements

Soumya Biswas is supported by the Bayer MEDHA Fellowship Program of Bayer India Foundation. Anupam Patra is recipient of Council of Scientific and Industrial Research- Senior Research Fellowship (CSIR-SRF). Gajraj Singh Kushwaha is supported by Indian Council of Medical Research (ICMR) Research Associate Award number BMI/11(45)/2020. We acknowledge Department of Biotechnology (DBT), Government of India (BT/INF/22/SP22660/2017) for ESRF Access Program for x-ray data collection. We acknowledge infrastructure support available through DBT-BUILDER program (BT/INF/22/SP42155/2021) at KIIT University.

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Author notes

  1. Prajita Paul

    Present address: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Authors and Affiliations

  1. School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT-DU), Bhubaneswar, 751024, India

    Soumya Biswas, Prajita Paul, Namrata Misra & Mrutyunjay Suar

  2. Transcription Regulation Group, International Centre of Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India

    Anupam Patra & Gajraj Singh Kushwaha

  3. KIIT-Technology Business Incubator, Kalinga Institute of Industrial Technology (KIIT-DU), Bhubaneswar, 751024, India

    Namrata Misra, Gajraj Singh Kushwaha & Mrutyunjay Suar

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Contributions

Soumya Biswas- Methodology, Investigation, Analysis, Data Curation, Validation, Writing - Original Draft, Anupam Patra- Investigation, Analysis, Data Curation, Validation, Prajita Paul- Investigation, Analysis, Data Curation, Validation, Namrata Misra- Analysis, Data Curation, Review & Editing Gajraj Singh Kushwaha- Conceptualization, Methodology, Validation, Supervision, Writing - Original Draft, Review & Editing, Mrutyunjay Suar- Supervision, Resources, Writing - Review & Editing, Project administration, Funding acquisition.

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Correspondence to Gajraj Singh Kushwaha or Mrutyunjay Suar.

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A patent application on KpFabI has been filed to Indian Patent office by Kalinga Institute of Industrial Technology (KIIT) Deemed to be University, Bhubaneswar.

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Biswas, S., Patra, A., Paul, P. et al. Structural and Biochemical Studies on Klebsiella Pneumoniae Enoyl-ACP Reductase (FabI) Suggest Flexible Substrate Binding Site. Protein J 43, 84–95 (2024). https://doi.org/10.1007/s10930-023-10176-8

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