Browsing by Author "Kaur, Navneet"

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Structural Diversity of D-Alanine: D-Alanine Ligase and Its Exploration in Development of Antibacterial Agents Against the Multi-Variant Bacterial Infections
(John Wiley and Sons Inc, 2022-04-07T00:00:00) Mayank; Sidhu, Jagpreet Singh; Joshi, Gaurav; Sindhu, Jayant; Kaur, Navneet; Singh, Narinder
D-alanine: D-alanine ligase (Rv2981c or Ddl) (EC 6.3.2.4) is a bacterial protein that performs critical functions for the proper growth and development of bacterial cells. Understanding the activity profile of Ddl within the various strains of the bacteria seems vital in broad-spectrum antimicrobial drug discovery. Therefore, to understand this heterologous nature, we focused on understanding the functional impact of the structural differences in the Ddl protein from Legionella pneumophila and E. coli bacteria. The structural features and dynamic behavior of Ddl, the interaction pattern, and the docking score of the Ddl-ATP/ADP are also found significantly different from each other. In-depth analysis viz molecular dynamics simulation and residue interaction network (RIN) studies provided us the detailed insight into the differences in the Ddl proteins from both the bacteria. In conclusion, understanding the inter-specific differences in the antibiotic targets Ddl in the case of diverse bacterial strains is vital for rationalizing the treatment of these infectious diseases. Therefore, the current work attempts to foresee the development of more efficacious antibacterial agents devoid of emerging resistance to bacterium strains. � 2022 Wiley-VCH GmbH.
Transgene-free genome editing supports the role of carotenoid cleavage dioxygenase 4 as a negative regulator of ?-carotene in banana
(Oxford University Press, 2022-02-01T00:00:00) Awasthi, Praveen; Khan, Shahirina; Lakhani, Hiralben; Chaturvedi, Siddhant; Shivani, S.; Kaur, Navneet; Singh, Jagdeep; Kesarwani, Atul Kumar; Tiwari, Siddharth
Carotenoid cleavage dioxygenases (CCDs) belong to a small gene family and have an important role in the intricate metabolism of carotenoids in plants. In this study we aimed to understand the regulatory mechanism of ?-carotene homeostasis by establishing transgene-free genome editing in banana. We found that the expression patterns of multiple CCDs were correlated with the concentrations of carotenoids in two cultivars with contrasting contents of ?-carotene, Nendran (high) and Rasthali (low). Higher expression of CCD4 in Rasthali (RAS-CCD4) was negatively correlated with ?-carotene accumulation in the fruit-pulp. Docking analysis and enzyme assays of purified RAS-CCD4 suggested that ?-carotene and 10-Apo-?-carotenal were the preferred substrates of RAS-CCD4. Bacterial complementation assays demonstrated the role of RAS-CCD4 in ?-carotene degradation, and this was further confirmed by in vivo overexpression of RAS-CCD4 in Arabidopsis, which resulted in significant reduction in ?-carotene concentration. CRISPR/Cas9-mediated editing of CCD4 was conducted in protoplasts and embryogenic cell lines of Rasthali, and carotenoid profiling in the resulting stable mutant lines showed greater increases in ?-carotene accumulation in the roots than in the leaves compared with unedited controls. The differences in expression of carotenoid pathway genes were correlated with differences in metabolites in the edited lines. Overall, our study suggests that carotenoid catabolism is regulated by CCD4 in ways that are tissue-and cultivar-specific, and it also demonstrates the successful use of the genome-editing tool in developing transgene-free biofortified lines of banana. � 2022 The Author(s) 2022.
Transgene-free genome editing supports the role of carotenoid cleavage dioxygenase 4 as a negative regulator of ?-carotene in banana
(Oxford University Press, 2022-02-01T00:00:00) Awasthi, Praveen; Khan, Shahirina; Lakhani, Hiralben; Chaturvedi, Siddhant; Shivani, S.; Kaur, Navneet; Singh, Jagdeep; Kesarwani, Atul Kumar; Tiwari, Siddharth
Carotenoid cleavage dioxygenases (CCDs) belong to a small gene family and have an important role in the intricate metabolism of carotenoids in plants. In this study we aimed to understand the regulatory mechanism of ?-carotene homeostasis by establishing transgene-free genome editing in banana. We found that the expression patterns of multiple CCDs were correlated with the concentrations of carotenoids in two cultivars with contrasting contents of ?-carotene, Nendran (high) and Rasthali (low). Higher expression of CCD4 in Rasthali (RAS-CCD4) was negatively correlated with ?-carotene accumulation in the fruit-pulp. Docking analysis and enzyme assays of purified RAS-CCD4 suggested that ?-carotene and 10-Apo-?-carotenal were the preferred substrates of RAS-CCD4. Bacterial complementation assays demonstrated the role of RAS-CCD4 in ?-carotene degradation, and this was further confirmed by in vivo overexpression of RAS-CCD4 in Arabidopsis, which resulted in significant reduction in ?-carotene concentration. CRISPR/Cas9-mediated editing of CCD4 was conducted in protoplasts and embryogenic cell lines of Rasthali, and carotenoid profiling in the resulting stable mutant lines showed greater increases in ?-carotene accumulation in the roots than in the leaves compared with unedited controls. The differences in expression of carotenoid pathway genes were correlated with differences in metabolites in the edited lines. Overall, our study suggests that carotenoid catabolism is regulated by CCD4 in ways that are tissue-and cultivar-specific, and it also demonstrates the successful use of the genome-editing tool in developing transgene-free biofortified lines of banana. � 2022 The Author(s) 2022.
Trends in small organic fluorescent scaffolds for detection of oxidoreductase
(Elsevier Ltd, 2021-06-15T00:00:00) Sidhu, Jagpreet Singh; Kaur, Navneet; Singh, Narinder
Oxidoreductases are diverse class of enzymes engaged in modulating the redox homeostasis and cellular signaling cascades. Abnormal expression of oxidoreductases including thioredoxin reductase, azoreductase, cytochrome oxidoreductase, tyrosinase and monoamine oxidase leads to the initiation of numerous disorders. Thus, enzymes are the promising biomarkers of the diseased cells and their accurate detection has utmost significance for clinical diagnosis. The detection method must be extremely selective, sensitive easy to use, long self-life, mass manufacturable and disposable. Fluorescence assay approach has been developed potential substitute to conventional techniques used in enzyme's quantification. The fluorescent probes possess excellent stability, high spatiotemporal ratio and reproducibility represent applications in real sample analysis. Therefore, the enzymatic transformations have been monitored by small activatable organic fluorescent probes. These probes are generally integrated with enzyme's substrate/inhibitors to improve their binding affinity toward the enzyme's catalytic site. As the recognition unit bio catalyzed, the signaling unit produces the readout signals and provides novel insights to understand the biochemical reactions for diagnosis and development of point of care devices. Several structural modifications are required in fluorogenic scaffolds to tune the selectivity for a particular enzyme. Hence, the fluorescent probes with their structural features and enzymatic reaction mechanism of oxidoreductase are the key points discussed in this review. The basic strategies to detect each enzyme are discussed. The selectivity, sensitivity and real-time applications are critically compared. The kinetic parameters and futuristic opportunities are present, which would be enormous benefits for chemists and biologists to understand the facts to design and develop unique fluorophore molecules for clinical applications. � 2021 Elsevier B.V.