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Expression, Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase

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Abstract

Patients in health-care settings develop nosocomial infections due to prolonged hospital stay. The Gram negative Klebsiella pneumoniae (K. pneumoniae), is a bacterial pathogen responsible for most nosocomial infections and are resistant to most current antibiotics. Hence, there is need for identification and validation of potential protein targets for design of new generation antibiotics. One of such targets is nicotinate nucleotide adenylyltransferase, an enzyme responsible for redox metabolism. This study focuses on novel expression, purification, and biophysical characterization of NNAT from K. pneumoniae. KpNNAT was over-expressed in T7 express™ Escherichia coli using the pGEX-4 T-1 expressions system and purified to > 98% homogeneity (~ 20 mg KpNNAT/g of the wet cell) using a combination of glutathione-agarose and immobilized Ni2+ affinity chromatography. KpNNAT indirectly showed “pseudo-specific activity” of 0.30 μmol/min/mg towards β-nicotinate mononucleotide and ATP using alcohol dehydrogenase as a secondary enzyme (in the presence of ethanol). Far-UV circular dichroism showed a ~ 38% predominantly alpha-helical and 16% β-strand secondary structural content. The binding of ATP to KpNNAT is entropically-driven with an overall ∆G° of ‒23.8 kJ/mol and dissociation constant of 69.1 µM. Data from this study suggest that KpNNAT can be expressed in E. coli, purified to homogeneity to yield high quantities of active recombinant enzyme for downstream biophysical studies such as X-ray crystallography.

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Acknowledgements

This work was supported by the South African Medical Research Council (SA-MRC) Self-Initiated Research Grant to IA and the South African Research Chairs Initiative (SARChI) of the Department of Science and Technology and National Research Foundation (grant 64788 to IA) and National Research Foundation National Equipment Programme (grant 88047 to Emeritus Prof HW Dirr). TD acknowledges the National Research Foundation (NRF) for funding her MSc degree. RM acknowledges the South African Department of Water and Sanitation for funding her MSc degree. OJ acknowledges the SA-MRC for bursary towards her MSc degree. CA also acknowledges the SARChI programme of the Department of Science and Technology and National Research Foundation for post-doctoral fellowship funding.

Funding

The work was funded by the South African Medical Research Council Self-Initiated Research Grant and the South African Research Chair Initiative of the Department of Science and Technology and National Research Foundation.

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Authors and Affiliations

  1. Protein Structure-Function and Research Unit, School of Molecular and Cell Biology, Faculty of Science, University of the Witwatersrand, Braamfontein, Johannesburg, 2050, South Africa

    Tasvi Daya, Olamide Jeje, Reabetswe Maake, Chinyere Aloke & Ikechukwu Achilonu

  2. Department of Biochemistry, School of Life Sciences, University of Kwa-Zulu Natal (Pietermaritzburg Campus), Scottsville, 3209, KwaZulu-Natal, South Africa

    Thandeka Khoza

  3. Department of Medical Biochemistry, Faculty of Basic Medical Sciences, Alex Ekwueme Federal University, Ndufu-Alike, Nigeria

    Chinyere Aloke

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  1. Tasvi Daya
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Contributions

TD, RM and OJ: Investigation, Methodology, Formal analysis, Writing—original draft. CA and TK: Review & editing of manuscript. IA: Writing and Review of manuscript, Supervision, Conceptualisation, Resources and Funding.

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Correspondence to Ikechukwu Achilonu.

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Ethics waiver for this study was awarded to Ikechukwu Achilonu by the Human Research Ethics (Medical) of the University of the Witwatersrand, Ref number: W-CBP-181009-01.

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Daya, T., Jeje, O., Maake, R. et al. Expression, Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase. Protein J 41, 141–156 (2022). https://doi.org/10.1007/s10930-021-10037-2

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