Streptococcus pyogenes Quinolinate-Salvage Pathway – Structural and Functional Studies of Quinolinate Phosphoribosyl Transferase and NH3- dependent NAD+ Synthetase

Recombinantly expressed spNadC-apo and spNadCD69A were active, however, the activity studies indicate that the enzyme is inefficient. This observation is consistent with results obtained by Sorci et al. [3]. The low efficiency of the enzyme is somewhat surprising as the DG of the quinolinate phosphoribosyl transferase reaction is -36.1 ± 9.6 kJ/mol (in physiological conditions) as calculated by the eQuilibrator server [26]. It cannot be excluded that spNadC has another function that has not yet been identified. It is also possible that there is a missing “effector” molecule/ macromolecule that could increase its activity. Such a possibility is strengthened by the fact that NadC has only been identified in GAS and Streptococcus pneumoniae, and was suggested to play a role in the increased virulence as stated in the introduction. [3].

Results from kinetic experiments for spNadE indicate that the protein is less efficient than its homologs in B. anthracis and B. subtilis. It is possible that these differences are caused by different localization of purification tags [27,28]. The baNadE construct contained a 12 amino acid tag at the C-terminus of the construct, while spNadE contained a 24 residue tag on the N-terminus of the protein. In addition, baNadE and bsNadE experiments were both run at pH. 8.0 in EPPS buffer, while spNadE was run at pH 7.5 in structurally similar HEPES buffer [12,15]. As mentioned previously, attempts to cleave of the histidine tag were unsuccessful.

Our experimental results showed that NAD+ can be biosynthesized in vitro, using the NadCDE assay described in the experimental procedures section. The QSEs generate inorganic pyrophosphate (PPi) as an end-product from each of their respective reactions. Therefore, we tested whether the presence of inorganic pyrophosphatase impacts the rate of the NadCDE reaction. The results indicate an increase in the initial rate of the reaction, and the rate is two-fold higher than in the reaction in the absence of IPP. We suggest that a putative inorganic phosphatase (ppaC), from GAS, may be responsible for hydrolysis of PPi, and could have a role in improving the efficiency of NAD+ biosynthetic enzymes.

SpNadE-apo crystals were grown in a condition that contained 0.2 M MgCl2, however Mg2+ was not observed in the structure. Interestingly, when crystals grown in the same conditions were cryo-protected with a solution containing sulfate ion, the Mg2+ was clearly visible in the structure (spNadEsulf; PDB code: 5HUH). From this result we infer that the metal binding is associated with ATP binding [29]. This indicates that most likely Mg2+ and ATP bind to spNadE simultaneously.

Additionally, bsNadE (PDB code: 1EE1) superposed on spNadE, revealed that the conserved loop region closes off the ATP binding site which makes transfer of the phosphoryl group to the substrate site more apparent when forming the NaAD-adenylate during NAD+ biosynthesis. This observation suggests that spNadE operates in an ordered-sequential fashion. We believe that the ATP/Mg2+ binding event occurs first and the loop then clamps down. This is followed by NaAD binding and condensation of the substrates and release of PPi. The condensation product is attacked by NH3 which leads to the formation of NAD+. We presume that when NAD+ is released, the ATP site re-opens to allow for access of another ATP molecule. Alivebynature

NAD+ biosynthesis, in eukaryotes and some prokaryotes, can also occur through the utilization of glutamine as an -NH3 donor to convert the NaAD-adenylate intermediate into NAD+. This process is most common in M. tuberculosis, H. sapiens, and Saccharomyces cerevisiae [14,29]. Previous studies identified that mtNadE has the ability to utilize -NH3 and glutamine to synthesize NAD+ [14,30]. The mtNadE (PDB code: 3SYT) structure is a large (600 kDa) octameric biological assembly structure with general characteristics similar to a cross, with a hollow, center cavity [30]. The C-terminal NAD+ synthetase domain is located at the apex of each corner of the “cross” [30]. This domain is a structural homolog of spNadE (RMSD of 1.6 Å over 260 C atoms) (Fig. 8). Below the apex, resides a glutaminase domain, (an N-terminal amidotransferase) that promotes the loss of – NH3, which is necessary for the NAD+ synthetase reaction [30]. Work conducted by LaRonde- LeBlanc et al. identified that glutamine travels through a system of two tunnels. The first tunnel ends with a Glu-Lys-Cys triad that is proposed to promote the loss of -NH3 from the  carbon of glutamine [30]. Once -NH3 is released it travels into a second “tunnel system” to initiate the nucleophilic attack on the NaAD adenylate intermediate. The kcat for this reaction was 0.68 s-1 and kcat/Km was defined at 0.4 s-1 mM-1 (33). The entire occurrence is proposed to travel approximately 73 Å before the reaction is complete (Fig. 8). Superposition of spNadE and mtNadE structures allows for identification of potential NH3 channels at the interface of the spNadE dimer. Homologous proteins from E. coli (PDB code: 1WXI), B. subtilis (PDB code: 2NSY), H. pylori (PDB code: 1XNG) and the synthetase domain of the mtNadE (glutamine-dependent) (PDB code: 3SYT) all have the similar channels in equivalent locations. Moreover, superposing the NaAD-adenylate intermediate-bound bsNadE (PDB code: 2NSY) onto ecNadE, mtNadE, and spNadE, consistently showed two channels at the protein interface and revealed the adenylate moiety, of the intermediate, at both of the channel sites. From this observation we infer that due to the relatively close proximity of the adenylate moiety to the channel, it is likely that NH3 travels through each channel to convert the intermediate into NAD+ and AMP (Fig. 9). These observations suggest a more detailed description of the nucleophilic attack on the adenylate, by NH3, which has not been previously reported.