
@article {123128,
	title = {Regulatory mechanisms of c-di-AMP synthase (MsDisA) protein from Mycobacterium smegmatis revealed by a structure - function analysis.},
	journal = {Protein Sci},
	year = {2023},
	month = {2023 Jan 20},
	pages = {e4568},
	abstract = {<p>Cyclic-di-nucleotide based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although, c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous B. subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Further, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of T. maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue. This article is protected by copyright. All rights reserved.</p>
},
	issn = {1469-896X},
	doi = {10.1002/pro.4568},
	author = {Gautam, Sudhanshu and Mahapa, Avisek and Yeramala, Lahari and Gandhi, Apoorv and Krishnan, Sushma and Vinothkumar, Kutti R and Chatterji, Dipankar}
}
@article {100624,
	title = {Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in  tracheoblasts.},
	journal = {Elife},
	volume = {10},
	year = {2021},
	month = {2021 Oct 08},
	abstract = {<p>Progenitors of the thoracic tracheal system of adult (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an HO generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/ Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of HO in minutes. The findings presented reveal that HO activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.</p>
},
	issn = {2050-084X},
	doi = {10.7554/eLife.68636},
	author = {Kizhedathu, Amrutha and Chhajed, Piyush and Yeramala, Lahari and Sain Basu, Deblina and Mukherjee, Tina and Vinothkumar, Kutti R and Guha, Arjun}
}
