Expression, Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase.

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 Ni2+ affinity chromatography. KpNNAT indirectly showed "pseudo-specific activity" of 0.30 µmol/min/mg towards ß-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% ß-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.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.