Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli.
Biogenesis of iron-sulfur (Fe-S) cluster-containing proteins relies on assistance of complex machineries. To date three systems, NIF, ISC, and SUF, were reported to allow maturation of Fe-S proteins. Here we report that the csdA-csdE (formally ygdK) genes of Escherichia coli constitute a sulfur-generating system referred to as CSD which also contributes to Fe-S biogenesis in vivo. This conclusion was reached by applying a thorough combination of both in vivo and in vitro strategies and techniques. Yeast two-hybrid analysis allowed us to show that CsdA and CsdE interact. Enzymology analysis showed that CsdA cysteine desulfurase activity is increased 2-fold in the presence of CsdE. Mass spectrometry analysis and site-directed mutagenesis showed that residue Cys-61 from CsdE acted as an acceptor site for sulfur provided by cysteine desulfurase activity of CsdA. Genetic investigations revealed that the csdA-csdE genes could act as multicopy suppressors of iscS mutation. Moreover, both in vitro and in vivo investigations pointed to a specific connection between the CSD system and quinolinate synthetase NadA.
Loiseau
Laurent
L
Laboratoire de Chimie Bactérienne, UPR-CNRS 9043, Institut de Biologie Structurale et Microbiologie, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Ollagnier-de Choudens
Sandrine
S
Lascoux
David
D
Forest
Eric
E
Fontecave
Marc
M
Barras
Frédéric
F
D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis*; D007700:Kinetics; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C022512:quinolinic acid synthetase; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase