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

Olga A. Esakova, Alexey Silakov, Tyler L. Grove, Douglas M. Warui, Neela Yennawar, Squire J. Booker

Research output: Contribution to journalArticle

Abstract

Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide 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 iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

Original languageEnglish (US)
Pages (from-to)14142-14151
Number of pages10
JournalJournal of the American Chemical Society
Volume141
Issue number36
DOIs
StatePublished - Sep 11 2019

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Dihydroxyacetone Phosphate
Quinolinic Acid
X ray crystallography
X Ray Crystallography
Phosphates
Catalysis
Molecules
Pyrococcus horikoshii
Iron
Oxaloacetic Acid
Ammonium Chloride
Crystals
Biosynthesis
Electron Spin Resonance Spectroscopy
Dehydration
Sulfur
Freezing
Hydroxyl Radical
NAD
Carrier concentration

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

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title = "An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase",
abstract = "Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide 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 iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, {"}intermediates{"} are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.",
author = "Esakova, {Olga A.} and Alexey Silakov and Grove, {Tyler L.} and Warui, {Douglas M.} and Neela Yennawar and Booker, {Squire J.}",
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T1 - An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase

AU - Esakova, Olga A.

AU - Silakov, Alexey

AU - Grove, Tyler L.

AU - Warui, Douglas M.

AU - Yennawar, Neela

AU - Booker, Squire J.

PY - 2019/9/11

Y1 - 2019/9/11

N2 - Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide 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 iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

AB - Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide 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 iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

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