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Structural and functional characterization of mycobacterial PhoH2 and identification of potential inhibitor of its enzymatic activity

  • Bacterial and Fungal Pathogenesis - Research Paper
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Abstract

Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as Km 4.873 ± 0.44 µM, Vmax 12.3817 ± 0.084 µM/min/mg, Kcat 0.0075 ± 0.00005 s−1, and Kcat/Km 0.0015 ± 0.000001 µM−1 s−1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation.

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Data availability

The authors agreed to the availability of data to ensure data transparency norms.

Code availability

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Abbreviations

TB:

Tuberculosis

M. tuberculosis :

Mycobacterium tuberculosis H37Rv

MDR-TB:

Multidrug-resistant TB

GTPases:

Guanosine triphosphatases

G-proteins:

GTP-binding proteins

SRP:

Signal recognition particle

PDB:

Protein Data Bank

RBC:

Real Biotech Corporation

LB agar:

Luria Bertani agar

LB broth:

Luria Bertani broth

IPTG:

Isopropyl β thiogalactoside

EDTA:

Ethylene diamine tetra-acetate

PBS:

Phosphate buffer saline

PMSF:

Phenylmethylsulphonyl fluoride

SDS PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

GST Resin:

Glutathione HiCap matrix

GEF:

Guanine nucleotide exchange factors

GAP:

GTPase-activating proteins

GDP:

Guanosine diphosphate

PCR:

Polymerase chain reaction

CD:

Circular dichoirism

UDP:

Uridine diphosphate

UTP:

Uridine triphosphate

CDP:

Cytidine diphosphate

CTP:

Cytidine triphosphate

ATP:

Adenosine triphosphate

ADP:

Adenosine diphosphate

Mt-PhoH2:

Mycobacterial PhoH2

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Acknowledgements

The authors thank Dr. Mary K. Ekka, CSIR-IGIB, for kindly providing inputs to the manuscript. They also acknowledge the research facilities provided by CSIR-IGIB.

Funding

The work is supported by Department of Science and Technology-SERB, Council of Scientific and Industrial Research-Institute of Genomics and Integrative Biology, under the research project GAP0145 (SERB-DST Grant no: EEQ/2016/000514).

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  1. Laxman S. Meena

    Present address: CSIR-Central Drug Research Institute, Lucknow, 226031, India

Authors and Affiliations

  1. CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India

    Shivangi , Yasmeen Khan, Mary Krishna Ekka & Laxman S. Meena

  2. Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC, Ghaziabad, Uttar Pradesh, 201 002, India

    Shivangi , Yasmeen Khan, Mary Krishna Ekka & Laxman S. Meena

  3. CSIR-Institute of Genomics and Integrative Biology, Academy of Scientific & Innovative Research (AcSIR), Mall Road, Delhi, 110007, India

    Laxman S. Meena

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Shivangi, Khan, Y., Ekka, M.K. et al. Structural and functional characterization of mycobacterial PhoH2 and identification of potential inhibitor of its enzymatic activity. Braz J Microbiol 55, 1033–1051 (2024). https://doi.org/10.1007/s42770-024-01267-4

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