Characterization of quinolinate synthases from Escherichia coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii indicates that [4Fe-4S] clusters are common cofactors throughout this class of enzymes.

Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor known primarily for its role as a co-substrate in a multitude of biological oxidation-reduction reactions that involve hydride transfers (1). It is also involved in myriad non-redox reactions such as adenylation, ADP-ribosylation, and histone deacetylation (2, 3), and in enzymes such as urocanase, in which its nicotinamide ring is proposed to form a transient covalent adduct with the substrate urocanate, facilitating catalysis via an electrophilic mechanism (4). Moreover, it serves as the initial substrate for the biosynthesis of thiamin thiazole in Saccharomyces cerevisiae (5). Quinolinic acid (QA) serves as the first common precursor to NAD and its derivatives in all organisms that synthesize NAD de novo; the subsequent steps that lead to NAD from QA are virtually identical across organisms (6). By contrast, there are two distinctly different pathways by which QA is formed. In most eukaryotes, QA is generated from the oxidative degradation of tryptophan in a series of five enzymatic steps, three of which involve molecular oxygen as a cosubstrate (6). The requirement for molecular oxygen in this pathway has led to its branding as the aerobic pathway for QA production, which distinguishes it from an alternative pathway (anaerobic pathway) found predominantly in prokaryotes, which does not display an obligate requirement for molecular oxygen. In the anaerobic pathway, l-aspartate is first converted to iminoaspartate (ImAsp) by l-aspartate oxidase (NadB); ImAsp is then condensed with dihydroxyacetone phosphate (DHAP) to form quinolinic acid in a reaction catalyzed by quinolinate synthase (NadA). The byproducts of the reaction are 2 mol of water and 1 mol of inorganic phosphate (Scheme 1) (7). Scheme 1 Reactions catalyzed by l-aspartate oxidase and quinolinate synthase NadB is a 60 kDa flavoprotein that contains one noncovalently bound molecule of flavin adenine dinucleotide (FAD) per polypeptide (8). During catalysis, l-aspartate is transiently oxidized to ImAsp with concomitant reduction of FAD to FADH2. Under oxic conditions, FADH2 is re-oxidized to FAD by molecular oxygen, with generation of hydrogen peroxide as a byproduct (Scheme 1). Under anoxic conditions, fumarate serves as the electron acceptor, being reduced to succinate (9). ImAsp is an unstable intermediate; it hydrolyzes to ammonia and oxaloacetate (OAA) at 37 °C and pH 8.0 with a half-life of 144 s (10). In vitro the NadB protein can be replaced by conducting the reaction in the presence of OAA and an ammonia source, such as ammonium sulfate (AS), which undergo Schiff-base formation to afford an equilibrium concentration of ImAsp (11). NadA from Escherichia coli has served as the prototype for the study of quinolinate synthases; however, detailed characterization and mechanistic analysis of the enzyme was originally hampered by its observed instability (10). The protein contains a CXXCXXC motif, which is found often in the primary structures of [4Fe-4S]-containing proteins of the ferredoxin class (12). This motif, as well as the enzyme’s reported tendency to lose catalytic competence in the presence of hyperbaric oxygen, led to the suggestion that E. coli NadA is an iron-sulfur (Fe/S) protein (13). This hypothesis was later confirmed. Analytical and spectroscopic studies by two independent laboratories showed that the protein binds and requires one [4Fe-4S]2+ cluster per polypeptide (14, 15). NadA has been purported to be a member of the Fe/S cluster-dependent class of the hydro-lyase family of enzymes (16), which includes aconitase, the prototype member (17). Aconitase contains a [4Fe-4S] cluster in which three of the iron atoms are ligated by three cysteine residues contributed by the protein scaffold. The fourth iron (Fea) provides a site for bidentate coordination by the substrate or product, with a corresponding change in its coordination geometry from tetrahedral to octahedral (18-21). Similarly, it is expected that the [4Fe-4S] cluster of NadA will also be ligated by three cysteinate protein ligands, allowing one iron site to be available for direct involvement in the reaction (16). The X-ray crystal structure of quinolinate synthase from the hyperthermophilic archaeon Pyrococcus horikoshii (Phk) was solved to 2.0 A in 2005. Surprisingly, the structure showed no evidence of an Fe/S cluster or any other type of cofactor, nor was mention of an Fe/S cluster made in the published report (22). Yet, the protein was stated to catalyze formation of QA with a specific activity of 2.2 μmol min-1 mg-1, an activity that is significantly higher than that reported for the Fe/S-containing E. coli enzyme (14). However, the structure contained three regions of poorly defined electron density, in which three cysteine residues that are strictly conserved among NadA proteins are found (Scheme 2). Herein, we show that the three corresponding cysteines in E. coli NadA are absolutely required for turnover in vitro, and most likely act as ligands to the [4Fe-4S] cluster. When C→S substitutions were made at each of the nine cysteine residues present in the primary structure of E. coli NadA, only substitutions at C113, C200, and C297 afforded inactive proteins when analyzed in their as-isolated (AI) or reconstituted (RCN) forms. Importantly, only C297 is present in the CXXCXXC motif (Scheme 2). In addition, we report the first published isolation of NadA from Mycobacterium tuberculosis (Mtb), and show that the protein contains a [4Fe-4S] cluster with similar spectroscopic parameters as that from E. coli. Unlike NadA from E. coli, the primary structure of the enzyme from Mtb does not contain a CXXCXXC motif, but does contain three conserved cysteine residues (Scheme 2). Last, we show that when Phk NadA is isolated under strict anaerobic conditions, it too contains a [4Fe-4S] cluster and displays a specific activity that is similar to that of the E. coli and Mtb enzymes. When it is overproduced recombinantly in the presence of o-phenanthroline, an Fe(II) chelator, or isolated aerobically, the protein is devoid of an Fe/S cluster and exhibits no catalytic activity. When this apo form of Phk NadA is reconstituted anaerobically with iron and sulfide, the [4Fe-4S] cluster reforms, and catalytic competence is regained. Since all annotated quinolinate synthases contain these three conserved cysteine residues, we argue that [4Fe-4S] clusters are common and requisite cofactors throughout this class of enzymes. Scheme 2 Relative positioning of cysteines in NadA proteins from P horikoshii, E. coli, and M. tuberculosisa