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.

Exploring NAD+ metabolism and NNAT: Insights from structure, function, and computational modeling.

Nicotinamide Adenine Dinucleotide (NAD+), a coenzyme, is ubiquitously distributed and serves crucial functions in diverse biological processes, encompassing redox reactions, energy metabolism, and cellular signalling. This review article explores the intricate realm of NAD + metabolism, with a particular emphasis on the complex relationship between its structure, function, and the pivotal enzyme, Nicotinate Nucleotide Adenylyltransferase (NNAT), also known as nicotinate mononucleotide adenylyltransferase (NaMNAT), in the process of its biosynthesis. Our findings indicate that NAD + biosynthesis in humans and bacteria occurs via the same de novo synthesis route and the pyridine ring salvage pathway. Maintaining NAD homeostasis in bacteria is imperative, as most bacterial species cannot get NAD+ from their surroundings. However, due to lower sequence identity and structurally distant relationship of bacteria, including E. faecium and K. pneumonia, to its human counterpart, inhibiting NNAT, an indispensable enzyme implicated in NAD + biosynthesis, is a viable alternative in curtailing infections orchestrated by E. faecium and K. pneumonia. By merging empirical and computational discoveries and connecting the intricate NAD + metabolism network with NNAT's crucial role, it becomes clear that the synergistic effect of these insights may lead to a more profound understanding of the coenzyme's function and its potential applications in the fields of therapeutics and biotechnology.

Obtaining high yield recombinant Enterococcus faecium nicotinate nucleotide adenylyltransferase for X-ray crystallography and biophysical studies.

Nicotinate nucleotide adenylyltransferase (NNAT) has been a significant research focus on druggable targets, given its indispensability in the biosynthesis of NAD+, which is crucial to the survival of bacterial pathogens. However, no information is available on the structure-function of Enterococcus faecium NNAT (EfNNAT). This study established the expression and purification protocol for obtaining a high-yield recombinant EfNNAT using the E. coli expression system and a single-step IMAC purification method. Approximately 101 mg of EfNNAT was obtained per 7.8 g of wet E. coli cells, estimated to be over 98 % pure. We further characterized the biophysical structure and determined the three-dimensional structure of the EfNNAT. Biophysical studies revealed a dimeric protein with a higher α-helical composition. The highly stable protein crystalizes in multiple conditions, yielding high-quality crystals diffracting between 1.78 and 2.80 Å. Two high-resolution crystal structures of EfNNAT in its native and adenine-bound forms were determined at 1.90 Å and 1.82 Å, respectively. The X-ray structures of the EfNNAT revealed the presence of phosphate and sulfate ions occupying and interacting with conserved amino acid residues within the putative substrate binding site, hence providing insight into the probable substrate preference of EfNNAT and, consequently, why EfNNAT may not prefer ß-nicotinamide mononucleotide as a substrate. With the accessibility to high-resolution structures of EfNNAT, further structural evaluation and drug-based screening can be achieved. Hence, we anticipate that this study will provide the basis for the discovery of structure-based inhibitors against this enzyme.

Combating Lassa Fever in West African Sub-Region: Progress, Challenges, and Future Perspectives.

Lassa fever (LF) is a rodent-borne disease that threatens human health in the sub-region of West Africa where the zoonotic host of Lassa virus (LASV) is predominant. Currently, treatment options for LF are limited and since no preventive vaccine is approved for its infectivity, there is a high mortality rate in endemic areas. This narrative review explores the transmission, pathogenicity of LASV, advances, and challenges of different treatment options. Our findings indicate that genetic diversity among the different strains of LASV and their ability to circumvent the immune system poses a critical challenge to the development of LASV vaccines/therapeutics. Thus, understanding the biochemistry, physiology and genetic polymorphism of LASV, mechanism of evading host immunity are essential for development of effective LASV vaccines/therapeutics to combat this lethal viral disease. The LASV nucleoprotein (NP) is a novel target for therapeutics as it functions significantly in several aspects of the viral life cycle. Consequently, LASV NP inhibitors could be employed as effective therapeutics as they will potentially inhibit LASV replication. Effective preventive control measures, vaccine development, target validation, and repurposing of existing drugs, such as ribavirin, using activity or in silico-based and computational bioinformatics, would aid in the development of novel drugs for LF management.

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.