Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid

Abstract

Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

“…Unlike eukaryotes, prokaryotes primarily synthesize QUIN from dihydroxyacetone phosphate and L-aspartate (4,5). The L-tryptophan-kynurenine pathway, however, is also found in a few bacteria (6,7), and vice versa, some eukaryotes are known to use the prokaryotic pathway (8)(9)(10).…”

An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase

“…In the previously studied PhNadA/QA complex, we observed a Hyperfine Sublevel Correlation (HYSCORE) signature of an 14 N nucleus of QA at a bonding distance to Fe a of the [4Fe-4S] + cluster ( Figure S3B) [19]. No such 14 N signals are observed in the reduced PhNadA/IA complex ( Figures S3C, S3E), suggesting that the amine group of IA is not bound to the Fe/S cluster.…”

“…To verify that the positioning of IA with respect to the open coordination site of the [4Fe-4S] cluster in crystallo is the same as that in solution, a study using continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies was carried out. X-band CW EPR spectra of the [4Fe-4S] + cluster in the reduced PhNadA/IA complex are significantly different from the spectrum of the substrate-free PhNadA control (g=[2.100, 1.937, 1.913]), the spectrum of the reduced PhNadA/DHAP complex (g=[2.076, 1.918, 1.904]) (see Figure 3), as well as the spectrum of the reduced PhNadA/QA complex [19]. Interestingly, the EPR spectrum of the PhNadA/IA complex consists of two contributions with different g-values (see simulated spectral components in Figure 3).…”

“…All general procedures have been previously described [19,22]. PhNadA and PhNadB were overproduced in E. coli and isolated as previously described [22].…”

“…A superposition of both PhNadA/IA and PhNadA/DHAP structures shows that both substrates occupy roughly the same position in the active site ( Figure S5A). A variant analysis of residues that coordinate to DHAP in the active site (His21Gln, His196Gln, and Ser126Ala) shows that none of the substitutions substantially alter the K M for DHAP in the PhNadA reaction [19].…”