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

Abstract

Streptococcus pyogenes, also known as Group A Strep (GAS), is an obligate human pathogen that is responsible for millions of infections and numerous deaths per year. Infection manifestations can range from simple, acute pharyngitis to more complex, necrotizing fasciitis. To date, most treatments for GAS infections involve the use of common antibiotics including tetracycline and clindamycin. Unfortunately, new strains have been identified that are resistant to these drugs therefore, new targets must be identified to treat drug resistant strains. This work is focused on the structural and functional characterization of three proteins: spNadC, spNadD, and spNadE. These enzymes are involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). The structures of spNadC and spNadE were determined. SpNadC is suggested to play a role in GAS virulence, while spNadE, functions as an NAD synthetase and is considered to be a new drug target. Determination of the spNadE structure uncovered a putative, NH₃ channel, which may provide insight into the mechanistic details of NH₃-dependent NAD⁺ synthetases in prokaryotes.

Graphical abstract

This work is focused on the structural and functional characterization of three proteins: spNadC, spNadD, and spNadE. These enzymes are involved in the biosynthesis of NAD⁺. The structures of spNadC and spNadE were determined. SpNadC is suggested to play a role in GAS virulence, while spNadE, functions as an NAD synthetase and is considered to be a new drug target.

INTRODUCTION

Streptococcus pyogenes, also known as group A strep (GAS), is a gram-positive bacterium responsible for infections associated with pharyngitis (“strep throat”), rheumatic fever, toxic-shock syndrome, and necrotizing fasciitis (“the flesh-eating” disorder) [1–3]. Severe cases of GAS diseases cause over 500,000 deaths globally which places GAS among the ten most common causes of death due to human pathogens [4]. During infection, non-specific exotoxin release from GAS promotes the activation of interferon-gamma (IFN-γ) [5]. IFN-γ aids in the initiation of tryptophan degradation by monocytes (blood)/macrophages (tissue) by way of the kynurenine pathway [5,6]. The end products of this pathway are quinolinic acid (QA) and picolinic acid [7]. It is presumed that tryptophan degradation is increased at the site of infection due to immune response. Subsequently, this increase leads to higher QA concentration at the site. GAS has evolved to utilize QA through the quinolinate- salvage pathway (QSP) which has the potential to enhance the virulence of the bacterium [3].

The QSP of GAS is responsible for the utilization of quinolinate in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). NAD⁺, is then reduced to the NADH form [3]. NAD⁺/NADH is a necessary cofactor in the progression of glycolysis, gluconeogenensis, the citric acid cycle, and the electron transport chain [8]. The QSP utilizes three proteins of interest: quinolinate phosphoribosyltansferase (spNadC), nicotinate mononucleotide transferase (spNadD), and a NH₃-dependent NAD⁺ synthetase (spNadE), all of which are responsible for the conversion of quinolinate, nicotinate, and nicotinamide into NAD⁺ (Fig. 1) [3]. The nadC gene has only been identified in GAS and Streptococcus pneumoniae and is suggested to play a role in increased virulence when compared to other streptococcal species [3].

Figure 1.

Integrated pathway for NAD⁺ biosynthesis in streptococcal species. This schematic diagram, adapted from Sorci et al., displays proteins and intermediates involved in NAD⁺ biosynthesis in all streptococcal species [3]. Virulent species of Streptococci, specifically S. pneumoniae and S. pyogenes, exhibit the ability to utilize quinolinate as an alternate route for nicotinate mononucleotide (NaMN) production which may provide an explanation for the increased virulence of these species [3].

In the QSP, spNadC is responsible for the first of three biosynthetic steps in the formation of NAD⁺ [3]. This enzyme is specifically responsible for the N-nucleoside bond formation between phosphoribosyl pyrophosphate (PRPP) and QA in order to form nicotinate mononucleotide (NaMN) (Fig. 2) [9]. GAS can also use nicotinate to synthesize NaMN by the action of nicotinic acid phosphoribosyltransferase (PncB) [3]. This pathway relies on niacin-transporting membrane proteins (NiaX) to import niacin and nicotinamide from the extracellular environment [3]. The specific means by which QA enters the cell has yet to be determined [3]. The ability to utilize various pathways for NAD⁺ biosynthesis most likely improves the survival of GAS during infection.

Figure 2.

Generalized schematic of the reactions catalyzed by QSEs. SpNadC and spNadE are the focal points of this manuscript.

SpNadD is responsible for the formation of a diphosphate ester in the conversion of NaMN and ATP into nicotinate adenine dinucleotide (NaAD) (Fig. 2) [3]. According to the Database for Essential Genes (DEG) and available literature, the expression of the nadD gene is integral for the survival of various pathogenic bacteria[3,10,11]. Moreover, the fact that the human homolog of NadD and spNadD have only 20% sequence identity makes the S. pyogenes protein an attractive target for development of antimicrobial compounds.

SpNadE is responsible for the formation of NAD⁺ by converting NaAD into NAD⁺ during the last step of the QSP (Fig. 2) [3]. This enzyme is relatively well studied and proteins from Bacillus anthracis, Bacillus subtilis, and Escherichia coli are functionally and structurally characterized. [12–14]. SpNadE is also thought to be an important protein for GAS survival and is considered to be a potential drug target [3].

This manuscript reports the first structures of quinolinate-salvage enzymes (QSEs) spNadC and spNadE, as well as biochemical and biophysical characterization of these enzymes.

RESULTS

Protein Isolation and Quaternary Structure Characterization

All proteins were isolated with high yields (at least 100 mg per liter of culture). Multiple attempts to cleave the purification tags, from each of the proteins, proved successful only for spNadD.

FPLC gel filtration produced peaks that correlated with the molecular weights of commercial protein calibration standards (GE Healthcare, Piscataway, NJ) at approximately 204 kDa for spNadC, 75 kDa for spNadD, and 65 kDa for spNadE. From this observation it was, assumed that the biological assembly for spNadC was hexameric, spNadD was trimeric, and spNadE was dimeric. Native gel results supported these initial assignments as well.

QSE dynamic light scattering (DLS) results were in agreement with gel filtration results. DLS experiments showed that the molecular weight of spNadC was approximately 212.3 (± 80.1) kDa, spNadD was reported at approximately 72 kDa (± 9.7), and spNadE also reported at 72 kDa (± 5.3). These results further support the findings that spNadC is a hexamer, spNadD is a trimer, and spNadE is a dimer.

SpNadCDE Activity Assay and QSE Complex Assumption

It was shown that spNadC, spNadD, and spNadE are able to produce NAD⁺. The reaction mixture containing all QSEs supplemented with MgCl₂, NH₄Cl, ATP, PRPP and QA was shown to produce NAD⁺. Moreover, we observed that the presence of inorganic pyrophosphatase (IPP) improves the rate of the reaction 2-fold. This observation, suggests that hydrolysis of pyrophosphate, which is a product in all reactions catalyzed by QSEs, may serve to drive NAD⁺ biosynthesis forward.

Additionally, these results prompted further investigation to determine whether the enzymes, in solution, operate as separate entities or as higher order macromolecular assemblies. Each of the isolated QSEs were tested on a native gel, to check whether such high order assemblies are formed. The results of these experiments clearly indicate that while all spNadC, spNadD and spNadE are present in solution in an oligomeric form, the formation of binary or tertiary complexes between these enzymes does not take place in the conditions used for the experiment.

SpNadE Activity

At increasing NaAD concentrations, spNadE exhibits Michaelis-Menten kinetics. With spNadE at 13 pmol concentration, results showed a K_m of 375 ± 5 μM. The k_cat was 4.2 ± 0.4 s⁻¹ and the k_cat/K_m was 11.09 s⁻¹ mM⁻¹. At the sample protein concentration, with increasing ATP, spNadE showed a K_m of 304 ± 4 μM, a k_cat of 5.6 ± 0.6 s⁻¹, and k_cat/K_m of 18.51 s⁻¹ mM⁻¹. When experimental concentrations greater than 4 mM ATP were tested, results indicated that excess ATP caused an inhibitory effect.

Kinetic results previously reported for Bacillus anthracis NadE (baNadE; 58.5% sequence identity) indicate apparent K_m values (K_m(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 K_m(app) of 179 μM (for NaAD) and 196 μM (for ATP) (12, 15).

SpNadC and spNadC_Δ69A Crystal Screening and Optimization Results

SpNadC-apo and spNadC_Δ69A (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_Δ69A 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 α/β hydrolase fold [16,17]. This fold commonly contains a central parallel β sheet that is composed of several beta strands which are flanked by α-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 α helical/8 β strand feature described above in the α/β 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 β sheet that is flanked on one side by four helices (Fig. 3).

Figure 3.

(A) Biological unit of spNadC. The hexameric assembly may be treated as a trimer of homodimers. The N- and C-terminal residues are marked as spheres. The terminal residues from protein chains forming the “top of the hexamer are labeled. (B) The dimeric section of the assembly is where the proposed active sites are located at the interface between the chains. One of the protein chains is colored to highlight secondary structure elements (α-helices – cyan, β-strands – magenta, loops – salmon).

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.

Table 1.

Summary of spNadC, spNadC_Δ69A, spNadE_sulf, and spNadE-apo data collection and structure refinement statistics. Values in parenthesis refer to the highest resolution shell.

Data Collection
Protein	spNadCΔ69A	spNadCΔ69A	spNadC	spNadEsulf	spNadE-apo
PDB accession code	5HUL	5HUO	5HUP	5HUH	5HUJ
Diffraction source	APS (21ID)	APS (22BM)	APS (19BM)	APS (21ID)	APS (19ID)
Wavelength (Å)	1.000	1.000	1.000	1.000	0.979
Space group	P23	C2221	C2221	P212121	P212121
a, b, c (Å)	179.6, 179.6, 179.6	106.2,186.3, 221.9	108.2, 189.0, 222.4	49.5, 92.1, 128.6	49.8, 93.0, 128.4
Resolution range (Å)	40–2.85 (2.95–2.85)	40–2.80 (2.85–2.80)	50.00–3.45 (3.46–3.40)	50.00–2.50 (2.54–2.50)	39.37–2.10 (2.14–2.10)
Total No. of reflections	44917 (2166)	54566 (2658)	26521 (1389)	19874 (856)	32708 (1586)
Completeness (%)	99.8 (99.5)	100 (100)	90.3 (95.2)	92.6 (82.9)	91.8 (90.5)
Redundancy	4.7 (4.6)	8.4 (8.5)	3.8 (3.9)	6.6 (5.3)	5.6 (5.0)
〈I/σ(I)〉	24.1 (2.0)	14.6 (2.3)	7.9 (1.8)	7.5 (2.4)	11.9 (2.3)
Rr.i.m.	0.129 (0.990)	0.162 (0.958)	0.140 (0.790)	0.159 (0.588)	0.156 (0.862)
Rp.i.m.	0.055 (0.434)	0.056 (0.331)	0.071 (0.389)	0.060 (0.238)	0.064 (0.373)
Overall B factor from Wilson plot (Å2)	84.6	64.6	45.3	26.8	29.6
Refinement
Resolution range (Å)	39.23–2.86 (2.92–2.85)	37.01–2.80 (2.87–2.80)	50–3.45 (3.54–3.45)	43.72–2.50 (2.56–2.50)	39.37–2.10 (2.15–2.10)
Completeness (%)	99.72 (99.6)	99.92 (100)	82.40 (72)	93.10 (92.1)	91.66 (89.5)
No. of reflections, working set	42638	51691	23687	18796	31023
No. of reflections, test set	2268	2768	1271	1338	1630
Final Rcryst	0.222 (0.301)	0.215 (0.317)	0.236 (0.298)	0.171 (0.222)	0.168 (0.204)
Final Rfree	0.246 (0.341)	0.255 (0.324)	0.290 (0.370)	0.225 (0.279)	0.212 (0.217)
RMS Deviations
Bonds (Å)	0.012	0.012	0.011	0.012	0.016
Angles (°)	1.2	1.5	1.6	1.4	1.6
Ramachandran Plot
Most favored (%)	96.6	96.7	93.5	98.8	99.2
Allowed (%)	100	99.0	99.5	99.8	99.6

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 Ų, as determined by PDBePISA [20]. The average area of the interfaces between the dimers, within the hexamer, is 805 Ų.

Figure 4.

(A) and (B) Putative active site of spNadC in overall and zoomed views. Residues forming the active site with two sulfate ions bound are shown in stick representation. (C) Complex between seNadC and QA (PDB code: 1QAP).

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 (S_N1) reaction on the δ⁺ C3 of PRPP to create the nucleoside bond forming NaMN and PP_i [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.

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.

Figure 5.

Two-dimensional projection of the CLANS clustering results of NadC homologous sequences. Proteins are indicated by dots. Lines indicate sequence similarity detectable with BLAST and are colored by a spectrum of shades of gray according to the BLAST P-value (black: P-value < 10–200, light grey: P-value < 10–6). Sequences corresponding to structures in PDB are indicated by blue dots, sequence of spNadC is indicated by red dot.

SpNadE Structure

The NadE structure bound to Mg²⁺ and SO₄²⁻ ions (spNadE_sulf) 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.

Figure 6.

(A) Cartoon representation of spNadE_sulf showing dimeric structure of the enzyme. Mg²⁺ is represented as a grey sphere. Sulfate ions are shown in stick representation. The N- and C-terminal residues, as well as residues marking the beginning and end of the disordered loops, are labeled. (B) Cartoon representation of bsNaE (PDB code: 1EE1) with marked ATP and NaAD binding sites (outlined by a box). (C) Putative spNadE active site residues are shown in green. NaAD (yellow) and the conserved loop (E215-A219; fuchsia) are modeled based on the structure of bsNadE. The residues are labeled according to spNadE sequence. The numbers in parentheses correspond to bsNadE sequence.

The spNadE_sulf 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 R_cryst value of 0.168 and an R_free 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 spNadE_sulf covers 2407 Ų.

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_α atoms) for stNadE; 0.6 Å (over 257 C_α atoms) for ecNadE, 0.7 Å (over 254 C_α atoms) for baNadE, and 0.9 Å (over 254 C_α atoms) for bsNadE [22].

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.

Sequence and structure analysis indicate the residues responsible for Mg²⁺ binding are the most highly conserved (100% sequence identity) between homologs. BLAST results for spNadE show Mg²⁺ binding residues include: S56, D60, E173, and T218 [18].

The ATP binding sites between the spNadE-apo and ecNadE AMP-bound structure (PDB code: 1WXI) do not show any significant cavity differences. However, a superposition of bsNadE (PDB code: 1EE1) on spNadE-apo illustrates the presumed location of the conserved loop region responsible for forming the completed active site (Fig. 6).

NaAD Binding Site

According to previous studies on similar −NH₃ dependent NadE homologs, NaAD was shown to bind across the dimeric chains in a cleft located below the ATP binding site [12]. This binding event should allow for the interaction of the γ-phosphate of ATP with the carboxyl group of the pyridine ring of NaAD in order to allow for the formation of the NaAD-adenylate intermediate. According to sequence similarity analysis, residues responsible for the formation and stabilization of the NaAD binding site, in spNadE, include: L53- S56, D60, S61, V89-L91, F140, G143, N144, A147, R148, R150, T168, E173, F178-K181, D184, T219, A220, L222, and A234 [18] (spNadE residues T219, A220, and L222 are not visible in the electron density). When compared to bsNadE, NaAD binding site residues have approximately 85% residue identity.

A comparison of spNadE-apo and NaAD-bound bsNadE revealed that the substrate binding pocket for bsNadE was visually larger than the predicted NaAD binding site for spNadE-apo. To confirm this finding the structures were superposed and the distances between F139 and M253 of bsNadE and F140 and K265 of spNadE were measured. When superposed, these residues were directly across from each other, on opposite sides of the binding pocket. These measurements were taken across the centermost point of the pocket in an attempt to confirm an actual size difference. More specifically, the distance between the ζ carbon of F128 and the δ sulfur of M253 of bsNadE was initially measured. Then, the distance between the ζ carbon of F140 and δ carbon K265 of spNadE-apo was measured. Results from these residue measurements identified that the distance between F128 to M253 in (bsNadE) was 1.8 Å wider than the distance between F140 and K265 (spNadE-apo). It was concluded that NaAD binding site is wider when the substrate is bound.

Moreover, further analysis of the area revealed that the NaAD binding site expansion seems to occur as an effect of the closure of the ATP binding site, after ATP is bound. This occurrence is presumed to be similar to how the compression of one end of a spring clamp causes expansion of the opposite end. In contrast, when NaAD is unbound, the ATP binding site expands. When NaAD is not bound, the structure reveals an open conformation of the ATP binding site and a narrow conformation NaAD site. It is apparent from this observation that the general mechanism for NAD⁺ biosynthesis is an ordered-sequential reaction [24].

Clustering Analysis of the NadE Homologs

The spNadE family (PF02540) comprises NH₃-dependent NAD⁺ synthetases, glutamine-dependent NAD⁺ synthetases, and GMP synthases (Fig. 7). GMP synthases (glutamine amidotransferases) form a large cluster, with slightly distinguishable internal subgroups of fungal and animal sequences. The dispersed sequences between the GMP synthase and NAD synthase clusters represent mostly bacterial and some archaeal uncharacterized proteins, some of which, based on sequence similarity, were annotated as “ExsB family protein”, “PP-loop family protein”, “LPS biosynthesis protein WbpG”. Bacterial glutamine-dependent NAD⁺ synthetases, represented by the structurally characterized protein from Mycobacterium tuberculosis (PDB code: 3SDB), form a distinct cluster, showing similarity to the above-mentioned dispersed sequences, while the eukaryotic glutamine-dependent NAD⁺ synthetases show higher similarity to NH₃-dependent NAD⁺ synthetases. NH₃-dependent NAD⁺ synthetases form three subclusters. Subclusters represented by the structurally characterized protein from S. avermitilis (PDB ID: 3N05) shows highest similarity to eukaryotic glutamine-dependent NAD⁺ synthetases and includes some sequences also annotated as glutamine-dependent NAD⁺ synthetases. The second subgroup is represented by NH₃-dependent NAD⁺ synthetases from H. pylori (PDB code: 1XNG) and C. jejuni (PDB code: 3P52). SpNadE is found in a small compact subcluster that also includes a number of other structurally characterized homologs (e.g. from B. anthracis (PDB code: 2PZB), Deinococcus radiodurans (PDB code: 4Q16), S. typhimurium (PDB code: 3HMQ), E. coli (PDB code: 1WXI)).

Figure 7.

Two-dimensional projection of the CLANS clustering results of NadE homologous sequences. Proteins are indicated by dots. Lines indicate sequence similarity detectable with BLAST and are colored by a spectrum of shades of gray according to the BLAST P-value (black: P-value < 10–200, light grey: P-value < 10–6). Sequences corresponding to structures in PDB are indicated by blue dots, sequence of spNadE is indicated by red dot.

DISCUSSION

We have identified that spNadC has a hexameric structure, similar in oligomeric structure to the human homolog (hsNadC). The hexamers may be treated as “trimers of dimers” where the active sites are located on the dimer interfaces. To date, the majority of the literature is consistent in the belief that the reaction occurs in an ordered-sequential fashion [9,25]. However, there is no agreement on the order in which the substrates bind. [9,25].

Recombinantly expressed spNadC-apo and spNadC_Δ69A 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 ΔG 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 (PP_i) 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 PP_i, 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 (spNadE_sulf; 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 PP_i. 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.

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 Cα 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 δ 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 k_cat for this reaction was 0.68 s⁻¹ and k_cat/K_m 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.

Figure 8.

The discovery of the possible direction of NH₃ entrance in spNadE. Surface representations of spNadE (yellow/blue) superposed on mtNadE tetramer (gray; PDB code: 3SYT) show the proposed direction of travel of NH₃ from the glutaminase/amidotransferase region to the NAD⁺ synthetase region. The tetramer of the 3SYT structure was used in order to better view the orientation of the superposed spNadE dimer with respect to the mtNadE NH₃ channel. The spNadE structure to the far right removes the superposed mtNadE structure to show evidence of two openings at the dimer interface of spNadE that may provide insight into the direction of nucleophilic attack on the NaAD-adenylate intermediate, directly behind that interface, during NAD⁺ biosynthesis.

Figure 9.

The proposed NH₃ passageway within the spNadE dimer. Cartoon representation of the NH₃ channel magnified at the proposed location of nucleophilic attack. NaAD and AMP molecules are superposed from mtNadE structure (PDB code: 3SYT). The NaAD and AMP end-products are located behind the NH₃ passageway (highlighted in pink). Cavities, indicated by the circles, could explain the direction of nucleophilic attack of NH₃. We presume that NH₃ enters through one of these cavities in order to promote the conversion of the NaAD adenylate intermediate into NAD⁺ and AMP.

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 6× 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 spNadC_Δ69A 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].

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 AKTA™ 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.

SpNadE Activity Assay

SpNadE activity assay was conducted in a final volume of 1 mL using a quartz cuvette following the protocol adapted from Nessi et al. [15]. Activity reaction solution concentrations were as follows: 60 mM HEPES (pH 7.5), 2 mM NaAD, 2 mM ATP, 10 mM ammonium chloride, 10 mM MgCl₂, and 20 mM KCl, 0.5 μg of spNadE, 1% ethanol and 1 μg of alcohol dehydrogenase (ADH; Sigma-Aldrich). Alcohol dehydrogenase was necessary to confirm NAD⁺ formation through its ability to promote a hydride shift to form NADH. NADH formation was detected by UV at 340 nm. Reactions were measured at 340 nm for 5 minutes. Kinetic experiments were prepared with increasing amounts of one substrate while keeping the other substrate at activity reaction concentrations. ATP kinetic experiments concentrations were as follows: 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, and 12 mM. NaAD kinetic experiment concentrations were as follows: 0.125 mM, 0.250 mM, 0.500 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM. Reactions were conducted using a Nanodrop 2000c UV-Vis spectrophotometer (Thermo Scientific; Waltman, MA). All experiments were conducted in triplicate, and the reported values are the averages of each cuvette experiment.

SpNadCDE Activity Assay

An assay was conducted to determine if the QSEs could produce NAD⁺, in vitro. The reaction was conducted at 1 mL volume in a quartz cuvette. The reaction solution contained: 50 mM HEPES (pH 7.5), 6 mM MgCl₂, 20 mM KCl, 10 mM NH₄Cl, 2 mM ATP, 0.2 mM phosphoribosyl pyrophosphate (PRPP), 0.36 mM quinolinic acid (QA), 2 μg bovine serum albumin (BSA), 1 % ethanol, 4 μg spNadC, 4 μg spNadD, 4 μg spNadE, 10 μg alcohol dehydrogenase (ADH). The activity assay was run for 30 min. In addition, the same assay was performed with the addition of 2 U of Thermostable IPP (New England Biolabs; Ipswich, MA).

Crystallization

All QSE crystal screening preparations were prepared, first, using the sitting-drop method. Initial screening utilized the, 96 well, Art Robbins Instruments (Sunnyvale, CA) Original Intelli-plate 96–2 plate. All of the drops were composed of a protein: well solution ratio of 1: 1.

Optimization experiments used both sitting and hanging drop techniques. Sitting-drop experiments used the 24-well Cryschem sitting-drop plate (Hampton Research, Aliso Viejo CA).

Data Collection and Processing

Prior to the diffraction experiments spNadC and spNadC_Δ69A crystals were cryo-cooled. Ten percent glycerol was used to cryo-protect the crystals grown from ammonium phosphate, and no cryoprotectant was used for other crystals. Data was collected at the Advanced Photon Source (APS) at Argonne National Laboratory (ANL, Lemont, IL). The data from the spNadC_Δ69A crystals were collected at Sector 22 belonging to the Southeast Regional Collaborative Access Team (SER-CAT); beamlines 22 BM and 22 ID. Data for spNadC-apo was collected at Sector 19 belonging to the Structural Biology Center (SBC); beamline 19 BM.

Prior to data collection spNadE_sulf crystal was transferred to a solution of cryo-protectant composed of 10 % glycerol, 5% w/v PEG 3350, and 0.1 M ammonium sulfate. The spNadE-apo crystal was cryo-cooled by direct transfer from the drop to liquid nitrogen. Diffraction data were collected at APS. The data from spNadE-apo crystal was collected at 19 ID of SBC and data from the spNadE_sulf crystal was collected at 21 ID-G at the Life Sciences Collaborative Access Team (LS-CAT). All data collection was performed at 100 K. Data processing was performed using HKL-2000 [34] and the details of the diffraction experiments are shown in Table 1.

Structure Determination and Validation

For each of the spNadC and spNadC_Δ69A structures HKL-3000 and MOLREP [34,35] were used for structure determination. Both of the spNadC_Δ69A structures were solved prior to the spNadC structure and molecular replacement was used for phasing. The P23 spNadC_Δ69A structure was determined first using the Homo sapiens NadC homolog (PDB code: 4KWV) as a starting model. The model of spNadC_Δ69A cubic form was used as a starting model for determination of the orthorhombic C222₁ form. Determination of the spNadC-apo structure was done by Fourier synthesis using the spNadC_Δ69A C222₁ structure to calculate phases. All structures were refined using HKL-3000 and Refmac [34,36]. Models were updated, refined and validated using Coot [22]. Final validation was done using Molprobity [37] and CheckMyMetal [38]). Summary information on model refinement and validation is included in Table 1.

HKL-3000 in combination with MOLREP were also used for determination of spNadE structures. Both of the spNadE structures were solved by molecular replacement. The structure of spNadE_sulf was solved first using the NAD⁺ synthetase Salmonella typhimurium structure (PDB code: 3HMQ) as the starting model. Refinement and validation was performed using the protocol described for spNadC. The structure of spNadE_sulf was used as a starting model for determination of the spNadE-apo structure. Information on model refinement and validation was summarized in Table 1.

Sequence Similarity Based Clustering

The sequences of the proteins corresponding to Pfam domains to which the analyzed proteins have been classified, were downloaded. In case of NadC, full length sequences corresponding to QRPTase_N (PF02749) and QRPTase_C (PF01729) were downloaded and merged, followed by removal of redundant sequences. Clustering of the sequences, to visualize sequence similarities between subgroups of related proteins, was performed based on their pair-wise BLAST similarity scores, using CLANS (CLuster ANalysis of Sequences) [39]. In CLANS the P-values of highly-scoring segment pairs (HSPs) obtained from an N × N BLAST search, are used to compute attractive and repulsive forces between each sequence pair. The clustering was performed using P-value threshold of 1e-6. A graphical representation of sequence families is achieved by moving the sequences according to the force vectors resulting from all pairwise interactions within the arbitrary distance space, and repeating the process until convergence.

Sequence analysis and sequence homology analysis results were obtained through the use of the NCBI Protein BLAST [40].

Various Computational Methods

The eQuilibrator server was used to calculate the predicted Gibbs free energy of the reactions of the QSEs [26]. The Dali server was used to identify structurally similar QSEs [41]). The SSM superpose command, in Coot, was used to show comparisons between analyzed structures [22]. PyMOL was used to visualize these superpositions and all figures involving protein structure were created using this software [42]. PDBePISA was used to calculate structural parameters of spNadC-apo, spNadC_Δ69A and spNadE [20]. The Consurf server was used to determine sequence conservation between similar proteins and created a conservation map on the surface of the structures [43].

Accession Numbers

All coordinates and structure factors were deposited to the Protein Data Bank (PDB) with the accession codes: 5HUL for spNadC_Δ69A - P23, 5HUO for spNadC_Δ69A - C222₁, 5HUP for spNadC-apo, 5HUH for spNadE_sulf, and 5HUJ for the spNadE-apo structure.

Abbreviations

ADH

alcohol dehydrogenase

ANL

Argonne National Laboratory

APS

Advanced Photon Source

BLAST

Basic Local Alignment Search Tool

BM

bending magnet beamline

BSA

bovine serum albumin

CLANS

CLuster ANalysis of Sequences

DEG

Database for Essential Genes

DLS

dynamic light scattering

GAS

Group A Streptococcus

GMP

guanidine monophosphate

HSPs

highly-scoring segment pairs

ID

insertion device beamline

IFN-γ

interferon-gamma

IPP

inorganic pyrophosphatase

LS-CAT

Life Sciences Collaborative Access Team

MOLREP

molecular replacement program

NaAD

nicotinate adenine dinucleotide

NAD⁺

nicotinamide adenine dinucleotide

NadC

quinolinate phosphoribosyltansferase

NadD

nicotinate mononucleotide transferase

NadE

NH₃-dependent NAD⁺ synthetase

NaMN

nicotinate mononucleotide

NiaX

niacin-transporting membrane proteins

OD₆₀₀

optical density at 600 nm wavelength

PDB

Protein Data Bank

PHT

phthalic acid

PncB

nicotinic acid phosphoribosyltransferase

ppaC

inorganic phosphatase

PPC

5-phosphoribosyl-1-(beta-methylene) pyrophosphate

PP_i

inorganic pyrophosphate

PRPP

phosphoribosyl pyrophosphate

QA

quinolinic acid

QRPTase_C

C-terminal domain quinolinate phosphoribosyl transferase

QRPTase_N

N-terminal domain quinolinate phosphoribosyl transferase

QSEs

quinolinate-salvage enzymes

QSP

quinolinate- salvage pathway

SBC-CAT

Structural Biology Center Collaborative Access Team

SER-CAT

Southeast Regional Collaborative Access Team