The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: investigation of cluster ligands |
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Authors: | Rousset Carine Fontecave Marc Ollagnier de Choudens Sandrine |
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Affiliation: | CEA, iRTSV, LCBM, 38054 Grenoble, France; CNRS, UMR5249, Grenoble, France; Université Joseph Fourier, Grenoble, France. |
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Abstract: | Nicotinamide adenine dinucleotide (NAD) derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate through the concerted action of l-aspartate oxidase and the [4Fe-4S] quinolinate synthase (NadA). Here, we addressed the question of the identity of the cluster ligands. We performed in vivo complementation experiments as well as enzymatic, spectroscopic and structural in vitro studies using wild-type vs. Cys-to-Ala mutated NadA proteins. These studies reveal that only three cysteine residues, the conserved Cys113, Cys200 and Cys297, are ligands of the cluster. This result is in contrast to the previous proposal that pointed the three cysteines of the C(291)XXC(294)XXC(297) motif. Interestingly, we demonstrated that Cys291 and Cys294 form a disulfide bridge and are important for activity. |
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Keywords: | Nicotinamide adenine dinucleotide Iron– sulfur cluster Site-directed mutagenesis Disulfide bridge Quinolinic acid Quinolinate synthase smCaps" >l-Aspartate oxidase |
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