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J Am Chem Soc. 2016 Sep 14;138(36):11802-9. doi: 10.1021/jacs.6b05884. Epub 2016 Aug 30.

Crystal Structures of Quinolinate Synthase in Complex with a Substrate Analogue, the Condensation Intermediate, and Substrate-Derived Product.

Author information

1
Metalloproteins Unit, Institut de Biologie Structurale, CEA, CNRS, Université Grenoble-Alpes , 71, Avenue des Martyrs, 38044 Grenoble Cedex 9, France.
2
CEA/DRF/BIG/CBM/BioCat, CNRS UMR 5249, LCBM, Université Grenoble Alpes, LCBM , Grenoble, France.

Abstract

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product. We also show that citrate can bind to both TmNadA K219R and its Y21F variant. The W structure indicates that condensation causes dephosphorylation. We propose that catalysis by the K219R/Y107F variant is arrested at the W intermediate because the mutated protein is unable to catalyze its aldo-keto isomerization and/or cyclization that ultimately lead to QA formation. Intriguingly, PGH binds to NadA with its phosphate group at the site where the carboxylate groups of W also bind. Our results shed significant light on the mechanism of the reaction catalyzed by NadA.

PMID:
27545412
DOI:
10.1021/jacs.6b05884
[Indexed for MEDLINE]

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