Expression, Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase.
Tasvi DayaOlamide JejeReabetswe MaakeChinyere AlokeThandeka KhozaIkechukwu AchilonuPublished in: The protein journal (2022)
Patients in health-care settings develop nosocomial infections due to prolonged hospital stay. The Gram negative Klebsiella pneumoniae (K. pneumoniae), is a bacterial pathogen responsible for most nosocomial infections and are resistant to most current antibiotics. Hence, there is need for identification and validation of potential protein targets for design of new generation antibiotics. One of such targets is nicotinate nucleotide adenylyltransferase, an enzyme responsible for redox metabolism. This study focuses on novel expression, purification, and biophysical characterization of NNAT from K. pneumoniae. KpNNAT was over-expressed in T7 express™ Escherichia coli using the pGEX-4 T-1 expressions system and purified to > 98% homogeneity (~ 20 mg KpNNAT/g of the wet cell) using a combination of glutathione-agarose and immobilized Ni 2+ affinity chromatography. KpNNAT indirectly showed "pseudo-specific activity" of 0.30 μmol/min/mg towards β-nicotinate mononucleotide and ATP using alcohol dehydrogenase as a secondary enzyme (in the presence of ethanol). Far-UV circular dichroism showed a ~ 38% predominantly alpha-helical and 16% β-strand secondary structural content. The binding of ATP to KpNNAT is entropically-driven with an overall ∆G° of ‒23.8 kJ/mol and dissociation constant of 69.1 µM. Data from this study suggest that KpNNAT can be expressed in E. coli, purified to homogeneity to yield high quantities of active recombinant enzyme for downstream biophysical studies such as X-ray crystallography.
Keyphrases
- klebsiella pneumoniae
- escherichia coli
- multidrug resistant
- gram negative
- healthcare
- acinetobacter baumannii
- drug resistant
- poor prognosis
- end stage renal disease
- binding protein
- chronic kidney disease
- newly diagnosed
- emergency department
- mass spectrometry
- high resolution
- prognostic factors
- machine learning
- resting state
- stem cells
- magnetic resonance imaging
- cell therapy
- biofilm formation
- functional connectivity
- staphylococcus aureus
- electron transfer
- social media
- long non coding rna
- ionic liquid
- patient reported outcomes
- alcohol consumption
- simultaneous determination
- bone marrow
- adverse drug
- patient reported