We propose that this event occurs, within each chain, with the assistance of highly conserved residues (T168, T180, K181, D184, D188, K269, R270) found on a loop structure (T168- D188) within the channel site. This suggests that NH3 access to the NAD-adenylate binding site is within this region. It is presumed that NH3 is transported with the assistance of the aforementioned conserved residues to initiate nucleophilic attack at the C20 (carbonyl carbon) of the intermediate. In summary, we infer that the NH3 channel in spNadE overlaps with the analogous channel present in the synthetase region of mtNadE.
Additionally, from surface representation comparisons with the bsNadE structure (PDB code: 1EE1), which contains an active site loop at the ATP site, we identified two other possible NH3 passageways that are close to the active site loop on each chain. The potential to inhibit the site for NH3 entrance could provide a very specific drug target that could have wide-ranging effects across multiple bacterial species that utilize NH3 for NAD+ biosynthesis.
EXPERIMENTAL PROCEDURES
Cloning and Protein Production – Genomic information on the QSEs were obtained from the Uniprot [31]. Synthetic genes with optimized codons were produced by DNA 2.0 (Menlo Park, CA) and introduced into vector pJexpress411 bearing kanamycin-resistance. Each of the QSEs were designed to contain an N-terminal purification tag (MHHHHHHSSGVDLGTENLYFQ↓SGSG). The purification tag includes a 6x His tag with a TEV-cleavage site (marked with an arrow). Each of the plasmids were introduced into competent BL21 (DE3) E. coli (New England Biolabs, Ipswich, MA). The plasmids were introduced using the heat/ cold shock method for bacterial transformation [32,33]. Upon transformation, the cells were grown on Luria Broth (LB) agar plates supplemented with kanamycin, for 12 hours at 37 °C. A single colony was grown in 5 mL of kanamycin-infused LB broth for 12-16 hours at 37 °C. After the 12-16 hour incubation, a portion of the cultures were stored in a 40 % glycerol/LB media solution and kept at -80 °C. During model building it was discovered that the synthesis of the nadC gene resulted in the omission of a codon and production of the spNadC69A deletion mutant. The gene was re-synthetized, to insert the alanine codon into the DNA sequence, by Genscript (Piscataway, NJ).
Overnight preparatory cultures of transformed BL21 (DE3) E. coli (New England Biolabs, Ipswich, MA) were transferred into 1 L of LB media and allowed to rotate at 250 RPM for approximately 2.5 hours at 37 °C. When an optical density (OD600) of 0.8 – 1.0 was reached 0.2 mM IPTG was added to promote protein over-expression as outlined by Sorci et al. [3]. After IPTG induction, culture temperature was dropped to 18 °C and allowed to rotate at 250 rpm for 12-16 hours [3]. Bioavailable NMN
Following incubation, cells were harvested by centrifugation for 20 min at 10,524 g at 4 °C using a Beckman Coulter Avanti J26S XPI centrifuge (Brea, CA). The cell pellet was resuspended in buffer containing: 500 mM NaCl, 150 mM Tris buffer (pH 7.5), 5 mM 2-mercaptoethanol, and protease inhibitors (Thermo Scientific, Waltham, MA). The cells were lysed using a Branson 450 Sonifier, and kept on ice to minimize protein denaturation. The cell lysate was separated into soluble and insoluble fractions using a Beckman Allegra X30 R centrifuge at 4 °C, at 3,849 g, for 20 minutes. The soluble fraction was decanted and centrifuged again using a Beckman Avanti J26S XPI centrifuge at 32,264 g for 20 min, at 4 °C. The soluble portion was then used for protein isolation by immobilized metal affinity chromatography using Ni-NTA resin (Qiagen, Hilden, DE). Protein was eluted from the column using increasing concentrations of imidazole in steps of 5 mM, 60 mM, 250 mM, and 500 mM. Purity of protein samples was confirmed by SDS-PAGE. The eluted protein solution was allowed to dialyze overnight, at 4°C, in buffer containing: 50 mM Tris, 150 mM NaCl, and 5 mM 2-mercaptoethanol. Finally, the dialyzed protein solution was further purified by size exclusion chromatography. All size exclusion experiments were conducted on an AKTATM Pure (General Electric, Piscataway, NJ) using a Superdex 200 HiLoad 16/60 column. Buffer conditions for these experiments were the same as the dialysis buffer. The fractions representative of the protein of interest were concentrated, divided into 1 mL aliquots, and stored at -80°C.
Dynamic Light Scattering. In preparation for experimentation, 2 mL of 2 mg/mL QSE was dialyzed for 12 hours in the dialysis solution described above. After dialysis the QSEs were diluted to 0.2 mg/mL, in dialysis buffer, and the sample was analyzed using the experimental procedure for instrumental analysis, as outlined in Malvern’s (Worcestershire, UK) Zetaseizer ZS90 protocol.
SpNadC Activity Assay: Activity reaction conditions, at a final volume of 1 mL, contained 50 mM HEPES (pH 7.5), 6 mM MgCl2, 200 μM PRPP, 300 μM QA, and 20 μg of NadC. The reaction time was 10 min per experiment. Reaction conditions for kinetic experiments which involved testing the effect of increasing PRPP on the activity of spNadC maintained the same activity assay concentrations, described above, with increasing amounts of PRPP at the following concentrations: 200 μM, 400 μM, 600 μM, 800 μM, 1 mM, 1.2 mM, 1.4 mM, and 1.6 mM. The above experiment was repeated with increasing amounts of QA at the following concentrations: 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM. Formation of NaMN was measured at 266 nm using a Nanodrop 2000c (Thermo Scientific, Waltman, MA). Each experiment was done in triplicate, at 1 mL, using a quartz cuvette.
