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

Allison H. Saunders, Amy E. Griffiths, Kyung Hoon Lee, Robert M. Cicchillo, Loretta Tu, Jeffrey A. Stromberg, Carsten Krebs, Squire J. Booker

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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 [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 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 shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.

Original languageEnglish (US)
Pages (from-to)10999-11012
Number of pages14
JournalBiochemistry
Volume47
Issue number41
DOIs
StatePublished - Oct 14 2008

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Pyrococcus horikoshii
Quinolinic Acid
Mycobacterium tuberculosis
Escherichia coli
Enzymes
L-aspartate oxidase
Biosynthesis
Ports and harbors
Sulfur
NAD
Cysteine
Proteins
Iron
Dihydroxyacetone Phosphate
Ferredoxins
Condensation reactions
Protein S
Electron Spin Resonance Spectroscopy
Bioinformatics
Computational Biology

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Saunders, Allison H. ; Griffiths, Amy E. ; Lee, Kyung Hoon ; Cicchillo, Robert M. ; Tu, Loretta ; Stromberg, Jeffrey A. ; Krebs, Carsten ; Booker, Squire J. / 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. In: Biochemistry. 2008 ; Vol. 47, No. 41. pp. 10999-11012.
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title = "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 M{\"o}ssbauer 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 {\AA} resolution [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 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 shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the {"}activity{"} reported does not correspond to quinolinate formation.",
author = "Saunders, {Allison H.} and Griffiths, {Amy E.} and Lee, {Kyung Hoon} and Cicchillo, {Robert M.} and Loretta Tu and Stromberg, {Jeffrey A.} and Carsten Krebs and Booker, {Squire J.}",
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pages = "10999--11012",
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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. / Saunders, Allison H.; Griffiths, Amy E.; Lee, Kyung Hoon; Cicchillo, Robert M.; Tu, Loretta; Stromberg, Jeffrey A.; Krebs, Carsten; Booker, Squire J.

In: Biochemistry, Vol. 47, No. 41, 14.10.2008, p. 10999-11012.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 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

AU - Saunders, Allison H.

AU - Griffiths, Amy E.

AU - Lee, Kyung Hoon

AU - Cicchillo, Robert M.

AU - Tu, Loretta

AU - Stromberg, Jeffrey A.

AU - Krebs, Carsten

AU - Booker, Squire J.

PY - 2008/10/14

Y1 - 2008/10/14

N2 - 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 [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 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 shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.

AB - 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 [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 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 shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.

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