Crystallographic Trapping of Reaction Intermediates in Quinolinic Acid Synthesis by NadA

Volbeda, Anne; Cabodevilla, Jaione Saez; Darnault, Claudine; Gigarel, Océane; Han, Thi-Hong-Liên; Renoux, Oriane; Hamelin, Olivier; Choudens, Sandrine Ollagnier de; Amara, Patricia

doi:10.1021/acschembio.7b01104

Cited by 9 publications

(42 citation statements)

References 36 publications

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“…18 Additional structures include the enzyme in complex with DHAP (which binds to Fe a via its C6 hydroxyl and C5 keto groups (numbering according to Figure 1), in complex with various analogues of IA, and in complex with potential intermediates. 18,20,21 Most recently, structures of NadA in complex with two potential reaction intermediates were reported, which supports the mechanism shown in Figure S1. 18,20 These intermediates include the condensation product between C1 of DHAP and C3 of IA (termed W), a resulting keto-aldo isomerization to yield intermediate X, and Schiff-base formation between N1 and the C3 aldehyde of formerly DHAP to yield intermediate Y (i.e., QP) (Figure S1).…”

Section: ■ Introductionsupporting

confidence: 78%

“…In the prevailing mechanism for NadA, a C3 carbanion of the enamine of IA attacks C4 of DHAP (see note on numbering in legend of Figure 1), eliminating orthophosphate and forming the nascent C-C bond of QA. Subsequent tautomerizations and Schiff-base formation between the amine of the formerly IA and the C6 aldehyde of the formerly DHAP lead to production of the QA precursor (QP), which has recently been renamed intermediate Y [9,18]. The C5 hydroxyl of the QP is proposed to ligate to Fe a to facilitate a dehydration reaction that is initiated by proton removal at C4 ( Figure S1) [17].…”

Section: Introductionmentioning

confidence: 99%

“…Subsequent tautomerizations and Schiff-base formation between the amine of the formerly IA and the C6 aldehyde of the formerly DHAP lead to production of the QA precursor (QP), which has recently been renamed intermediate Y. 9,18 The C5 hydroxyl of the QP is proposed to ligate to Fe a to facilitate a dehydration reaction that is initiated by proton removal at C4 (Figure S1). 17 Recently, a number of structures of NadAs from multiple organisms in the presence of substrates, substrate analogues, products, and potential intermediates have been published.…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2019

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Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD +) 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 non-cysteinyl-ligated iron ion (Fe a), 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 Fe a 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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Section: ■ Introductionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2019

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“…A complex, resulting in the structure 6F6Z, formed by soaking mTS-N 4 -OH-dCMP with meTHF solution, was obtained in search of TS inactivation intermediate steps preceding that observed within the 4EZ8 structure (mTS ternary complex with the covalently bound inhibitor and non-covalently bound DHF; Scheme 1 ). A similar approach has proven successful in studies involving several groups of enzymes [ 28 , 29 , 30 , 31 , 32 , 33 ]. The N 4 -OH-dCMP molecule in the resulting complex is most likely not bound to the catalytic cysteine residue, thus being probably in the same conformation as the inhibitor molecule present in the binary mTS-N 4 -OH-dCMP complex structures (PDB ID: 4EIN and 4PSG).…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies

Maj

Jarmuła

Wilk

et al. 2021

IJMS

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Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.

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“…High-resolution structures of PhNadA with bound QA, maleate, and citraconate (pdb codes 5kto, 5ktr, and 5kts) 15 have unveiled the formation of a second active site cavity that we named cavity II. 10 We reasoned that a transient additional 13 to the TmNadA* open form (pdb code 4p3x). 12 The three domains and the C-terminal region of TmNadA*Y21F are sequentially colored cyan, green, and hazel brown; TmNadA* is shown in gray.…”

Section: ■ Introductionmentioning

confidence: 99%

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA

et al. 2021

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Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

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Crystallographic Trapping of Reaction Intermediates in Quinolinic Acid Synthesis by NadA

Cited by 9 publications

References 36 publications

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

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

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA

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