The effect of Mg2+ and Ni2+ on the kinetics and thermodynamics of ATP-binding, catalysis, and stability of Klebsiella pneumoniae nicotinate nucleotide adenylyltransferase

Abstract

Hospital-acquired infections (HAIs) have become a burden to the healthcare system with a global impact of 19.1% and 7.5% in developing and developed nations, respectively. Bacteria associated with HAIs are referred to as ESKAPE pathogens. They are multidrug resistant and Klebsiella pneumoniae is one of those pathogens. The bacterium has also shown to inherit heat resistance from a heat shock protein. Thus, becoming a double threat. The therapeutic approach to combat HAIs would be to target NAD+ synthesis because it is vital in the survival of prokaryotes. The focus would be to inhibit nicotinate nucleotide adenylyltransferase (NNAT) which is essential in NAD+ formation. Inhibiting the enzyme would decrease the NAD+ pool and eliminate the bacterium. NNATs are metal binding proteins and have selective preference for metal ions. Mg2+ has been the preferred metal ion and in some species Ni2+ being the favoured metal ion. The aim was to assess the influence of Mg2+ or Ni2+ on the structure, kinetics and ATP binding. The objectives are to express and purify KpNNAT with a pET expression system and nickel affinity chromatography, followed by functional characterisation with an ADH-mediated dual assay. Structural characterisation with far-UV circular dichroism (cd), fluorescence spectroscopy and size exclusion-high performance liquid chromatography. Thermostability with far-UV cd and thermal shift assay with a SYPRO orange dye. Isothermal titration calorimetry to assess ATP binding and obtain kinetics parameters. The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpNNAT. The protein has 0.09 µmole·min-1 ·mg-1 increase in pseudo specific activity in the presence of Mg 2+ and maximal activity compared to Ni2+. Far-UV cd suggest that the protein is α-helical in the presence of Mg2+ while there is observed loss of α-helical content in the presence of Ni2+. The protein is thermally stable in the presence of Mg2+ as opposed to Ni2+. The protein adopts a compact conformation in the presence of Mg2+ . Fluorescence studies suggest that Mg2+ creates a polar environment in the hydrophobic pocket for ATP binding. While a hydrophobic environment is promoted in the presence of Ni2+ . ATP binding is weak in the presence of Mg2+ and no binding is observed in the presence of Ni2+. NMN binds with a low affinity in the presence of Mg2+, thus suggesting that it is not the preferred mononucleotide substrate. Furthermore, in the absence of cations and presence of Ni2+ the reaction was endothermic and in the presence of Mg2+ the reaction is exothermic, entropically favourable, and spontaneous. To conclude, the difference in the activity, structure and binding in the presence of metal ion suggests that Mg2+ is the preferred divalent metal ion for IV KpNNAT catalysis. Therefore, this study contributes to better understanding the enzyme to subsequently improve administered antibiotics for HAIs.