Abstract
A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction.
Highlights
From the ‡Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, and the §Institute for Health Science, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
Tion reaction between L-aspartate and dihydroxyacetone phosphate (DHAP)1 that is catalyzed by two enzymes, L-aspartate oxidase (LAO; nadB gene product), and quinolinate synthase (QS; nadA gene product)
LAO catalyzes the oxidation of Laspartate to iminoaspartate, while QS catalyzes the condensation of iminoaspartate with DHAP to produce quinolinate (QA), which is converted to NAD via a metabolic sequence common to all organisms
Summary
A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. Some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. We observed the presence of LAO in Pyrococcus horikoshii, an anaerobic hyperthermophilic archaeon [8]. This is the first example of the occurrence of LAO either in the Archaea or in obligate anaerobic organisms. The genes that encode the homologue of all other enzymes involved in the de novo NAD biosynthesis were identified in the P. horikoshii genome. The crystal structure of QS from P. horikoshii was determined in the presence of malate, a substrate analogue of iminoaspartate. Knowledge of the structure of QS should yield information about the catalytic mechanism of the enzyme
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