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

Abstract

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of L-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mössbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 Å resolution [H. Sakuraba, H. Tsuge, K. Yoneda, N. Katunuma, and T. Ohshima, (2005) J. Biol. Chem. 280, pp. 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/ S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 μmol min -1 mg -1 , significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 μmol min -1 mg -1 ), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products

The kynurenine pathway and the brain: Challenges, controversies and promises

“…For example, bacteria can neo-synthesize quinolinic acid (Marinoni et al, 2008; Reichmann et al, 2015; Sakuraba et al, 2005; Saunders et al, 2008), and unique prokaryotic enzymes can degrade kynurenic acid (Taniuchi and Hayaishi, 1963). Bacterial neosynthesis of quinolinic acid begins with the condensation of iminoaspartate with dihydroxyacetone, and the latter compound also inhibits α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) (Garavaglia et al, 2009).…”

The Crystal Structure of Fe4S4 Quinolinate Synthase Unravels an Enzymatic Dehydration Mechanism That Uses Tyrosine and a Hydrolase-Type Triad

“…both catalyze a dehydration process and coordinate a Fe 4 S 4 cluster with only three cysteine protein ligands, which in NadA are organized in a CX86 -112CX 86-99C motif. [5][6][7] Consequently, it was suggested that the unique iron site of the NadA Fe 4 S 4 cluster should also be directly involved in dehydration steps, acting as a Lewis acid. Partial mechanistic information has been obtained from the two available crystal structures of NadA, 8 , 9 which correspond to the inactive apo form of the enzyme.…”

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

“…monodentate or bidentate coordination of the substrate) (47). The interacting iron atom could facilitate nucleophilic attack at the sulfate sulfur by acting as a Lewis acid, analogous to aconitase (29).…”