Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster

Shukla, Ekta; Thorat, Leena; Bhavnani, Varsha; Bendre, Ameya D.; Pal, Jayanta K.; Nath, Bimalendu B.; Gaikwad, Sushama M.

doi:10.1016/j.ijbiomac.2016.06.097

Cited by 24 publications

(25 citation statements)

References 39 publications

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“…Px_EclTre displayed optimum activity at pH 5 ( Fig. 1D), similar to the characteristic feature of acidic trehalases (pH 5-7) [6,[10][11][12][13]. The optimum temperature for Px_EclTre activity was found to be 55°C ( Fig.…”

Section: Resultssupporting

confidence: 58%

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae

et al. 2019

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Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from E. cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (±0.05) mm, kcat of 6254.72 min−1 and Vmax 0.2 (±0.002) mm·min−1 at 55 °C and acidic pH. Crystal structures of Px_EclTre were determined in the ligand‐free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand‐free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate‐binding site. A cluster of five tryptophans lined the −1 substrate‐binding subsite, interacted with crucial active site residues and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.

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Section: Resultssupporting

confidence: 58%

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae

et al. 2019

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“…In this study, we selected the prokaryotic soluble expression system pCold-TF to prepare recombinant feIFN-ωa and feIFN-ωb. The pCold-TF system is a highly efficient soluble expression system with a His-tagged TF that allows the expressed protein to be effectively modified, folded, and secreted into the cytoplasm [43,44]. Following the construction of recombinant E. coli strains and induction by IPTG, the rfeIFN-ωa/ωb proteins were expressed in their soluble forms and analyzed via SDS-PAGE.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-ω Proteins

Wang

Han

et al. 2020

Viruses

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Cats are becoming more popular as household companions and pets, forming close relationships with humans. Although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. Interferons (IFNs), especially type I IFNs (IFN-α, IFN-β, and interferon omega (IFN-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. Nevertheless, there is limited knowledge regarding feline IFN-ω (feIFN-ω), compared to IFN-α and IFN-β. In this study, we cloned the genes encoding feIFN-ωa and feIFN-ωb from cat spleen lymphocytes. Homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feIFN-ω. The recombinant feIFN-ωa and feIFN-ωb proteins were expressed in their soluble forms in Escherichia coli, followed by purification. Both proteins exhibited effective anti-vesicular stomatitis virus (VSV) activity in Vero, F81 (feline kidney cell), Madin-Darby bovine kidney (MDBK), Madin-Darby canine kidney (MDCK), and porcine kidney (PK-15) cells, showing broader cross-species antiviral activity than the INTERCAT IFN antiviral drug. Furthermore, the recombinant feIFN-ωa and feIFN-ωb proteins demonstrated antiviral activity against VSV, feline coronavirus (FCoV), canine parvovirus (CPV), bovine viral diarrhea virus (BVDV), and porcine epidemic diarrhea virus (PEDV), indicating better broad-spectrum antiviral activity than the INTERCAT IFN. The two novel feIFN-ω proteins (feIFN-ωa and feIFN-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats.

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“…A longstanding biochemical adjustment of survival under dry conditions, is the ability of desiccation-responsive synthesis and accumulation of biomolecules including trehalose, mannitol, glycerol, Heat-Shock (HS) and Late Embryonic Abundant (LEA) proteins, proline, glycine-betaine, gamma aminobutyric acid, alanine, and glucosamine (Crowe and Madin, 1974; Tunnacliffe and Lapinski, 2003; Yoder et al, 2006; Kikawada et al, 2008; Philip et al, 2008; Benoit et al, 2009; Mitsumasu et al, 2010; Thorat et al, 2012; Hidalgo et al, 2014; Shukla et al, 2015, 2016, 2018; Yoshida et al, 2016; Thorat et al, 2017; Mazin et al, 2018). These compatible solutes not only offer protection to the drying tissues but also trigger various signaling responses during recovery.…”

Section: Desiccation Tolerance Strategies In Insectsmentioning

confidence: 99%

Insects With Survival Kits for Desiccation Tolerance Under Extreme Water Deficits

Thorat

Nath

2018

Front. Physiol.

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The year 2002 marked the tercentenary of Antonie van Leeuwenhoek’s discovery of desiccation tolerance in animals. This remarkable phenomenon to sustain ‘life’ in the absence of water can be revived upon return of hydrating conditions. Today, coping with climate change-related factors, especially temperature-humidity imbalance, is a global challenge. Under such adverse circumstances, desiccation tolerance remains a prime mechanism of several plants and a few animals to escape the hostile consequences of fluctuating hydroperiodicity patterns in their habitats. Among small animals, insects have demonstrated impressive resilience to dehydration and thrive under physiological water deficits without compromising on revival and survival upon rehydration. The focus of this review is to compile research insights on insect desiccation tolerance, gathered over the past several decades from numerous laboratories worldwide working on different insect groups. We provide a comparative overview of species-specific behavioral changes, adjustments in physiological biochemistry and cellular and molecular mechanisms as few of the noteworthy desiccation-responsive survival kits in insects. Finally, we highlight the role of insects as potential mechanistic models in tracking global warming which will form the basis for translational research to mitigate periods of climatic uncertainty predicted for the future.

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Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster

Cited by 24 publications

References 39 publications

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae

Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-ω Proteins

Insects With Survival Kits for Desiccation Tolerance Under Extreme Water Deficits

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