Functional Studies of Quinolinate Phosphoribosyl Transferase and NH3- dependent NAD+ Synthetase

Kinetic results previously reported for Bacillus anthracis NadE (baNadE; 58.5% sequence identity) indicate apparent Km values (Km(app)) for NaAD and ATP as 152 μM and 289 μM, respectively [12,15]. Kinetic data reported previously for the Bacillus subtilis NAD synthetase (bsNadE; 56.7% sequence identity) reported a Km(app) of 179 μM (for NaAD) and 196 μM (for ATP) (12, 15).

SpNadC and spNadC69A Crystal Screening and Optimization Results – SpNadC-apo and spNadC69A (unintentionally generated deletion mutant – see the Material and Methods section) crystals were identified within 48 hours of crystallization setup. Crystals were identified in a variety of conditions through the use of the Index Screen (Hampton Research, Aliso Viejo CA) and Wizard Classic I & II Screens (Rigaku, Bainbridge Island, WA). While crystal formation was fast and plentiful, obtaining crystals that diffracted to a resolution higher than 4.0 Å was challenging. Despite the fact that crystals appeared in various conditions, their cubed morphology was the same.

Diffraction data for structure determination of spNadC69A were the first obtained from crystals grown in the following conditions: (1) 1M ammonium phosphate, 0.1 M sodium citrate tribasic/citric acid (pH 5.5), 0.2 M sodium chloride (Wizard Classic II, #33, Rigaku Bainbridge Island, WA), and (2) 20% w/v PEG 6000, 0.1 M BIS-TRIS (pH 6.4), 0.48 ammonium sulfate, 1.25 mM sodium bromide.

Diffraction results from the wild-type spNadC were obtained from crystals grown in 0.48 M ammonium sulfate, 25% w/v PEG 6000 and 0.1 M BIS-TRIS (pH 6.5).

SpNadE Crystallization – SpNadE crystals were produced from: 0.1 M Tris (pH 8.5), 20% w/v PEG 4000, and 0.2 M magnesium chloride. In hanging and sitting drop preparations, small needle- like clusters of crystals were obtained within 24 hours. While, these crystals were not suitable for diffraction, it was later found that beneath the needle clusters, within the same condition, were thin, rectangular, plate-like crystals. These crystals diffracted well, yet appeared inconsistently.

SpNadC Structure – According to the Pfam database, spNadC monomer is a part of a family of proteins that have an / hydrolase fold [16,17]. This fold commonly contains a central parallel  sheet that is composed of several beta strands which are flanked by -helices. SpNadC also belongs to the TIM-phosphate binding superfamily of proteins that are grouped by the presence of a TIM- barrel structure containing the same 8  helical/ 8  strand feature described above in the / hydrolase family [18,19]. The barrel-type fold corresponds to the C-terminal part of spNadC, while the N-terminal part of the protein contains an anti-parallel  sheet that is flanked on one side by four helices (Fig. 3).

The crystal structure obtained using condition 1 belongs to the cubic system and P23 space group, while condition 2 lead to orthorhombic crystals with C2221 symmetry. There were four chains in the asymmetric unit for the P23 structure, and six in the C2221 structure. For most of the protein chains, residues 6-288 were visible in the density. Data collection and processing details as well as structure solution and refinement details are outlined in Table 1.

All determined spNadC structures revealed the presence of hexameric assemblies in the crystals. This is in agreement with gel filtration, dynamic light scattering, and native gel results which indicate that spNadC is a hexamer in solution. It is assumed that the hexamer observed in the crystal structure is the same hexamer found in solution. The identified oligomeric structure may be treated as a “trimer of dimers” (Fig. 4). The proposed substrate binding site is formed by two protein chains that compose the dimer. The area of interaction between chains forming the dimer is very large and is calculated to be 2623 Å2, as determined by PDBePISA [20]. The average area of the interfaces between the dimers, within the hexamer, is 805 Å2. Buy NMN

SpNadC Active Site – Structural comparisons of spNadC with a homolog from Salmonella enterica (seNadC; PDB code: 1QAP; sequence identity 41%) bound to QA indicates that residues T139, R140, K141, H162, and R163 of spNadC are identical with the residues of the seNadC structure. (Fig. 4). A comparison of the Mycobacterium tuberculosis homolog (mtNadC; PDB code: 1QPR; sequence identity 40%) bound to substrate analogs, phthalic acid (PHT) (QA analog) and 5- phosphoribosyl-1-(beta-methylene) pyrophosphate (PPC; a PRPP analog) to spNadC also indicates that the residues involved in QA and PRPP binding are highly conserved not only in terms of sequence, but also in their structure. Sharma et al. suggested that PRPP is bound to two Mg2+ ions which are considered responsible for stabilizing the pyrophosphate region of PRPP [21]. However, to date there have been no structures determined with PRPP bound.

In mtNadC residues R105, R139 and R162 (which correspond to R106, R140 and R163 in spNadC) are suggested to be responsible for binding QA while D173 and R48 (D174 and K49 in spNadC) along with a series of coordinated water molecules bind to the phosphate group and the metal ion on the “pyrophosphate side” of PRPP. In mtNadC, the nitrogen atom from the backbone of G249, the carbonyl oxygen from A268, the nitrogen atom from G270, and the side chain of H274 are proposed to be involved in binding of the PRPP C5 phosphate group [21]. No mechanistic details for this reaction are available, but it is presumed that the nitrogen of the pyridine ring of QA initiates a nucleophilic substitution (SN1) reaction on the + C3 of PRPP to create the nucleoside bond forming NaMN and PPi [21]. Structural comparisons between spNadC and the homologs reported to the PDB also suggests that the QA and PRPP binding sites are created from residues from one chain of the dimer and not from the interaction of both chains together. Superposition of structures of homologous enzymes from Homo sapiens, Salmonella enterica, and Mycobacterium tuberculosis also indicates very similar substrate binding locations.