Monthly Archives: July 2018

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

Clustering Analysis of the NadC Homologs – Clustering classification of the sequences was carried out to visualize groups of more similar sequences and similarities between them. SpNadC is composed of two domains: a quinolinate phosphoribosyl transferase, N-terminal domain (QRPTase_N (PF02749)), and a quinolinate phosphoribosyl transferase, C-terminal domain (QRPTase_C (PF01729)). Therefore, in order to perform sequence similarity based clustering of its homologs, full length sequences of proteins from both Pfam families were downloaded and merged, followed by the removal of redundant sequences.

Nicotinate-nucleotide pyrophosphorylases show high internal similarity, with dispersed bacterial and archaeal sequences, where the subgroup of molybdenum utilization proteins (ModD) can be distinguished. Eukaryotic nicotinate-nucleotide pyrophosphorylases form a more clearly separated group (Fig. 5). The group of nicotinate phosphoribosyltransferases, represented by structurally characterized proteins from Thermoplasma acidophilum (PDB code: 1YTD) and Pyrococcus furiosus (PDB code: 2I14) is clearly isolated and detaches from all the other sequences when more stringent P- values are applied.

SpNadE Structure – The NadE structure bound to Mg2+ and SO42- ions (spNadEsulf) was the first of the NadE structures to be determined (Fig. 6). The protein crystallized in primitive orthorhombic system and P212121 space group with two protein chains in the asymmetric unit. The structure was determined at 2.5 Å resolution. The model of the structure includes a dimer and the protein chains include residues 9-214 and 229- 282. Residues 215-228 from the highly conserved active site loop, are missing in both structures, which indicates that they are disordered in the absence of substrates. Similarly, the polyhistidine tags were also not modeled, as there is no visible electron density that corresponds to this fragment of the molecule.

The spNadEsulf structure was used as a starting model during the determination of the spNadE- apo structure. The spNadE-apo structure was determined at 2.1 Å resolution, and the final model was refined to Rcryst value of 0.168 and an Rfree of 0.212. The model included residues 9-96, 98-215, 228- 282 for each of the protein chains that form the dimer, present in the asymmetric unit. According to PDBePISA calculations, the dimer interface for spNadE-apo and the same interface for spNadEsulf covers 2407 Å2.

The single chain of spNadE has primarily alpha helical character with eleven alpha helices, three parallel beta strands and two 310 helices. The sequence and structure based searches revealed that there are several spNadE homologs that have their structure experimentally determined. Homologous NAD+ synthetases that had the highest sequence identity originated from S. typhimurium (PDB code: 3HMQ, 68 % sequence identity; stNadE), E. coli (PDB code: 1WXI, 65% sequence identity; ecNadE), B. anthracis (PDB code: 2PZA; 58% sequence identity; baNadE) and B. subtilis (PDB code: 2NSY; 57% sequence identity; bsNadE). The structure of these proteins were used to identify the putative substrate binding sites for spNadE, and gave the following RMSD values, using Coot SSM superpose: 0.7 Å (over 251 C atoms) for stNadE; 0.6 Å (over 257 C atoms) for ecNadE, 0.7 Å (over 254 C atoms) for baNadE, and 0.9 Å (over 254 C atoms) for bsNadE [22]. Sublingual NMN

spNadE Active Site – Sequence similarity analysis in combination with structural studies of structural homologs bsNadE and ecNadE, indicates that there are approximately 18 residues responsible for the architecture of the active site. The closed conformation of the active site loop observed in structure of bsNadE (PDB code: 1EE1) is not present in the spNadE structures reported here. This result is possibly due to high entropy in that region. Through sequence similarity studies of this region, specifically residues 215-223, it was determined that these residues are highly conserved and make up a loop structure within that region. The loop was modeled using the structure of a homolog – bsNadE (PDB code: 1EE1) as a template (Fig. 6). It should be noted that the bsNadE (PDB code: 1EE1) structure was one of the few structures that determined the orientation for the loop region [23]. This loop has also been indicated in the stabilization of Mg2+ within the ATP binding site, as the ATP binding site shares three residues with this site. For spNadE, the residues of the loop of interest include (corresponding bsNadE residues are in bold): E215 (L204), K216 (K205), V217 (E206), P218 (P207), T219 (T208), A220 (A209), D221 (D210), L222 (L211), E223 (L212). Both loops have approximately 67% sequence identity. Unfortunately, numerous attempts to co-crystallize spNadE

with ATP, NaAD, and/or NAD+, with and without Mg2+, were unsuccessful.
spNadE ATP/ Mg2+ Binding – ATP and Mg2+ binding sites in spNadE were shown to be

composed of residues from a single protein chain. Moreover, structural analysis indicates that the binding of ATP occurs solely within the individual chains of the dimeric assembly and not at the interface. Residues involved in the conserved “SGGXD” motif (residues S56-D60) are also found at this site.

Sequence similarity studies of spNadE, using homologs from ecNadE (PDB code: 1WXI) and bsNadE (PDB code: 1EE1), identified residues: L53, G54, I55, S56, D60, S61, V89, R90, L91, R150, T168, K197, V217, P218, and T219 to be responsible for providing a binding pocket for ATP. Residues P218 and T219 (as well as any other residues between 216-227) are not visible in the electron density due to conformational flexibility in that region.