Abstract
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.
Highlights
IntroductionThe spike in the rate of nosocomial infection caused by multi-drug resistant (MDR)
Understanding the biophysical structure of an enzyme is paramount to its novel application and the design of inhibitors with therapeutic potential
We have comprehensively described the biophysical features of recombinant KpNNAT in relation to the effect of divalent cations on the structure and function of this essential enzyme
Summary
The spike in the rate of nosocomial infection caused by multi-drug resistant (MDR). As identified by Rice (2008), a group of MDR strains known as ESKAPE pathogens, consisting of both Gram-positive and negative species (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), are the predominant cause of nosocomial infections [1]. These pathogens are notorious for their ability to effectively escape the biocidal action of antibacterial drugs; they are 4.0/). About 7–14% of nosocomial infections in ICU and 3–20% of neonatal septicemia are caused by K. pneumoniae
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