8AII

High Resolution Crystal Structure of Enterococcus faecium Nicotinate Nucleotide Adenylyltransferase Complexed with Adenine

Summary for 8AII

• Entry DOI: 10.2210/pdb8aii/pdb
• Descriptor: Probable nicotinate-nucleotide adenylyltransferase, SULFATE ION, ADENINE, ... (5 entities in total)
• Functional Keywords: enterococcus, faecium, nicotinate, nucleotide, adenylyltransferase, adenine, transferase
• Biological source: Enterococcus faecium
• Total number of polymer chains: 1
• Total formula weight: 24983.72

Authors

Pandian, R.,Jeje, O.A.,Sayed, Y.,Achilonu, I.A. (deposition date: 2022-07-26, release date: 2023-08-16, Last modification date: 2023-08-23)

Primary citation

Jeje, O.,Pandian, R.,Sayed, Y.,Achilonu, I.
Obtaining high yield recombinant Enterococcus faecium nicotinate nucleotide adenylyltransferase for X-ray crystallography and biophysical studies.
Int.J.Biol.Macromol., 250:126066-126066, 2023

PubMed Abstract: 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 β-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.

PubMed: 37544558
DOI: 10.1016/j.ijbiomac.2023.126066

Experimental method

X-RAY DIFFRACTION (1.82 Å)