Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P)+ biosynthesis

doi:10.1016/0003-9861(91)90270-S

Archives of Biochemistry and Biophysics

Volume 284, Issue 1, January 1991, Pages 106-111

https://doi.org/10.1016/0003-9861(91)90270-S Get rights and content

Abstract

The ability of niacin to relieve the growth-inhibiting effect of hyperoxia on Escherichia coli can be attributed to the dioxygen sensitivity of quinolinate synthetase. The activity of this enzyme within E. coli was diminished by exposure of the cells to 4.2 atm O₂, while the activity in extracts was rapidly decreased by 0.2 atm O₂. Neither catalase nor superoxide dismutase afforded detectable protection against the inactivating effect of O₂, indicating that H₂O₂ and O₂⁻ were not significant intermediates in this process. Nevertheless, H₂O₂ at 1.0 mm did inactivate quinolinate synthetase, even under anaerobic conditions and in the absence of catalatic activity which might have generated O₂. Addition of paraquat to aerobic cultures of E. coli caused an inactivation of quinolinate synthetase, which may be explained in terms of an increase in the production of H₂O₂. The O₂-dependent inactivation of quinolinate synthetase in extracts was gradually reversed during anaerobic incubation and this reactivation was blocked by α,α′-dipyridyl or by 1,10-phenanthroline. The sequence of the quinolinate synthetase “A” protein contains a—cys-w-x-cys-y-z-cys—sequence, which is characteristic of (FeS)₄-containing proteins. This sequence, together with the effect of the Fe(II)-chelating agents, suggests that the O₂-sensitive site of quinolinate synthetase is an iron-sulfur cluster which is essential for the dehydration reaction catalyzed by the A protein.

References (25)

O.R. Brown et al.
Biochem. Biophys. Res. Commun
(1979)
O.R. Brown et al.
Biochem. Biophys. Res. Commun
(1980)
M. Heitkamp et al.
Biochim. Biophys. Acta
(1981)
P.R. Gardner et al.
Free Rad. Biol. Med
(1990)
O.R. Brown et al.
Free Rad. Biol. Med
(1990)
S. Nasu et al.
J. Biol. Chem
(1982)
C.F. Kuo et al.
J. Biol. Chem
(1987)
K.T. Yasunobu et al.
W.R. Waud et al.
Arch. Biochem. Biophys
(1975)
J.M. McCord et al.
J. Biol. Chem
(1969)

R.F. Beers et al.

J. Biol. Chem

(1952)

N. Suzuki et al.

Biochim. Biophys. Acta

(1973)

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Nicotinamide adenine dinucleotide (NAD) derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate through the concerted action of l-aspartate oxidase and the [4Fe–4S] quinolinate synthase (NadA). Here, we addressed the question of the identity of the cluster ligands. We performed in vivo complementation experiments as well as enzymatic, spectroscopic and structural in vitro studies using wild-type vs. Cys-to-Ala mutated NadA proteins. These studies reveal that only three cysteine residues, the conserved Cys113, Cys200 and Cys297, are ligands of the cluster. This result is in contrast to the previous proposal that pointed the three cysteines of the C₂₉₁XXC₂₉₄XXC₂₉₇ motif. Interestingly, we demonstrated that Cys291 and Cys294 form a disulfide bridge and are important for activity.
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Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox biological reactions. NAD derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate (DHAP) as the result of the concerted action of two enzymes, l-aspartate oxidase (NadB) and quinolinate synthetase (NadA). We report here the characterization of NadA protein from E. coli. When anaerobically purified, the isolated soluble protein contains 3–3.5 iron and 3–3.5 sulfide/polypeptide chain. Mössbauer spectra of the ⁵⁷Fe-protein revealed that the majority of the iron is in the form of a (4Fe–4S)²⁺ cluster. An enzymatic assay for quinolinate synthetase activity was set up and allowed to demonstrate that the cluster is absolutely required for NadA activity. Exposure to air leads to degradation of the cluster and inactivate enzyme.
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Nicotinic acid requirement points to a defect in NAD biosynthesis. Nicotinic acid, although not a natural precursor of NAD, can by-pass the early steps of NAD biosynthesis, by entering the pathway immediately after the step catalyzed by quinolinate synthase A (NadA), a [4Fe–4S] enzyme (Gardner and Fridovich, 1991; Draczynska-Lusiak and Brown, 1992; Ollagnier-de Choudens et al., 2005). Thus, the NAD biosynthesis defects exhibited by icsS mutant could arise from an IscS- dependence of NadA for acquiring its Fe/S cluster.
Owing to the versatile electronic properties of iron and sulfur, iron sulfur (Fe/S) clusters are perfectly suited for sensing changes in environmental conditions and regulating protein properties accordingly. Fe/S proteins have been recruited in a wide array of diverse biological processes, including electron transfer chains, metabolic pathways and gene regulatory circuits. Chemistry has revealed the great diversity of Fe/S clusters occuring in proteins. The question now is to understand how iron and sulfur come together to form Fe/S clusters and how these clusters are subsequently inserted into apoproteins. Iron, sulfide and reducing conditions were found to be sufficient for successful maturation of many apoproteins in vitro, opening the possibility that insertion might be a spontaneous event. However, as in many other biological pathways such as protein folding, genetic analyses revealed that Fe/S cluster biogenesis and insertion depend in vivo upon auxiliary proteins. This was brought to light by studies on Azotobacter vinelandii nitrogenase, which, in particular, led to the concept of scaffold proteins, the role of which would be to allow transient assembly of Fe/S cluster. These studies paved the way toward the identification of the ISC and SUF systems, subjects of the present review that allow Fe/S cluster assembly into apoproteins of most organisms. Despite the recent discovery of the SUF and ISC systems, remarkable progress has been made in our understanding of their molecular composition and biochemical mechanisms. Such a rapid increase in our knowledge arose from a convergent interest from researchers engaged in unrelated fields and whose complementary expertise covered most experimental approaches used in biology. Also, the high conservation of ISC and SUF systems throughout a wide array of organisms helped cross-feeding between studies. The ISC system is conserved in eubacteria and most eukaryotes, while the SUF system arises in eubacteria, archaea, plants and parasites. ISC and SUF systems share a common core function made of a cysteine desulfurase, which acts as a sulfur donor, and scaffold proteins, which act as sulfur and iron acceptors. The ISC and SUF systems also exhibit important differences. In particular, the ISC system includes an Hsp70/Hsp40-like pair of chaperones, while the SUF system involves an unorthodox ATP-binding cassette (ABC)-like component. The role of these two sets of ATP-hydrolyzing proteins in Fe/S cluster biogenesis remains unclear. Both systems are likely to target overlapping sets of apoproteins. However, regulation and phenotypic studies in E. coli, which synthesizes both types of systems, leads us to envisage ISC as the house-keeping one that functions under normal laboratory conditions, while the SUF system appears to be required in harsh environmental conditions such as oxidative stress and iron starvation. In Saccharomyces cerevisiae, the ISC system is located in the mitochondria and its function is necessary for maturation of both mitochondrial and cytosolic Fe/S proteins. Here, we attempt to provide the first comprehensive review of the ISC and SUF systems since their discovery in the mid and late 1990s. Most emphasis is put on E. coli and S. cerevisiae models with reference to other organisms when their analysis provided us with information of particular significance. We aim at covering information made available on each Isc and Suf component by the different experimental approaches, including physiology, gene regulation, genetics, enzymology, biophysics and structural biology. It is our hope that this parallel coverage will facilitate the identification of both similarities and specificities of ISC and SUF systems.

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^☆: This work was supported by research grants from the National Science Foundation; the American Cancer Society; the Council for Tobacco Research, U.S.A., Inc.; and the National Institutes of Health.

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Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P)+ biosynthesis☆

Abstract

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochim. Biophys. Acta

Free Rad. Biol. Med

Free Rad. Biol. Med

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

J. Biol. Chem

J. Biol. Chem

Biochim. Biophys. Acta

Quinolinate synthetase: The oxygen-sensitive site of de novo NAD(P)⁺ biosynthesis☆