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

Autor: Shivangi; CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India.; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC, Ghaziabad, Uttar Pradesh, 201 002, India., Khan Y; CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India.; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC, Ghaziabad, Uttar Pradesh, 201 002, India., Ekka MK; CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India.; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC, Ghaziabad, Uttar Pradesh, 201 002, India., Meena LS; CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India. laxmans.meena@cdri.res.in.; Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC, Ghaziabad, Uttar Pradesh, 201 002, India. laxmans.meena@cdri.res.in.; CSIR-Central Drug Research Institute, Lucknow, 226031, India. laxmans.meena@cdri.res.in.; CSIR-Institute of Genomics and Integrative Biology, Academy of Scientific & Innovative Research (AcSIR), Mall Road, Delhi, 110007, India. laxmans.meena@cdri.res.in.
Jazyk: angličtina
Zdroj: Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] [Braz J Microbiol] 2024 Jun; Vol. 55 (2), pp. 1033-1051. Date of Electronic Publication: 2024 Feb 22.
DOI: 10.1007/s42770-024-01267-4
Abstrakt: 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 Mg 2+ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as K m 4.873 ± 0.44 µM, V max 12.3817 ± 0.084 µM/min/mg, K cat 0.0075 ± 0.00005 s -1 , and K cat /K m 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.
(© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)
Databáze: MEDLINE
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