Abstract
Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.
Highlights
Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems
The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet
Purification of SufS and SufE—E. chrysanthemi SufS-His6 protein was overproduced in E. coli BL21(DE3) transformed with the plasmid pET-Shis
Summary
Materials—All chemicals were of reagent grade and obtained from Sigma-Aldrich Chemical Co. or Fluka unless otherwise stated. E. coli strains were grown aerobically at 37 °C in Luria-Bertani (LB) rich medium [32]. E. chrysanthemi strains were grown aerobically in LB medium at 30 °C. The pEG202 and pJG4 –5 vectors were used to express Suf proteins fused to the DNAbinding protein LexA and to the transcriptional activation motif B42, respectively. The sufE and sufS inserts were amplified using E. chrysanthemi chromosomal DNA as a template and primer 6s/6as and 5s/5as, respectively. The sufE and sufS inserts were digested by EcoRIXhoI and cloned into the EcoRI-XhoI-digested pEG202 and pJG4 –5, yielding the pB42 and pLexA plasmid series: pB42-E, pB42-S, pLexA-E, pLexA-S. The ha-sufS and c-myc-sufE inserts were obtained by PCR amplification using pB42-S or pB42-E as templates, and oligonucleotides NcoHA/5as and Nco-c-Myc-sufE/6as, respectively. The ha-sufS and cmyc-sufE PCR products were NcoI/XhoI digested and inserted into
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