The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: investigation of cluster ligands.
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.
Rousset
Carine
C
Commissariat à l'Energie Atomique, iRTSV, LCBM, 38054 Grenoble, France.
Fontecave
Marc
M
Ollagnier de Choudens
Sandrine
S
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry*
D004220:Disulfides; D029968:Escherichia coli Proteins; D008024:Ligands; D013455:Sulfur; D007501:Iron; D019883:Alkyl and Aryl Transferases; C532144:quinolinate synthase, E coli; D003545:Cysteine