Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg 2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg 2+ . Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg 2+ , while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg 2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg 2+ . Conclusively, the presence of Mg 2+ induces a conformation in KpPrAT that favours nucleotide binding.
Keyphrases
- klebsiella pneumoniae
- binding protein
- escherichia coli
- multidrug resistant
- healthcare
- oxidative stress
- protein protein
- amino acid
- poor prognosis
- high resolution
- emergency department
- dna damage
- dna binding
- case report
- molecular dynamics simulations
- small molecule
- adverse drug
- acute care
- gold nanoparticles
- social media
- health insurance
- ischemia reperfusion injury
- computed tomography
- induced apoptosis
- liquid chromatography
- endoplasmic reticulum stress
- human health
- high performance liquid chromatography
- metal organic framework