IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

Abstract

SynopsisIscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/ SufA paralogs blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthesis pathway in E. coli cells under aerobic conditions and that addition of the ironbound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the cluster assembly in protein ThiC of the thiamin biosynthesis pathway, aconitase B of the citrate acid cycle, and endonuclease III of the DNA base excision repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin, and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogs are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions. Keywords aconitase; branched-chain amino acids; dihydroxyacid dehydratase; iron-sulfur clusters; IscA/SufA paralogs; thiamin IntroductionIron-sulfur clusters are one of the most ancient and ubiquitous redox centers in biology. They are involved in diverse physiological processes including respiratory electron transfer, nitrogen fixation, photosynthesis, biosynthesis of amino acids, thiamin, heme, biotin, and lipoic acid, DNA synthesis and repair, RNA modification, and the regulation of gene expression [1,2]. However, the mechanism underlying the iron-sulfur cluster assembly is still not fully understood [3]. The discovery of cysteine desulfurase (NifS) Experimental Mutant strains and cell growthThe E. coli deletion mutants in which iscA and sufA were in-frame deleted were previously constructed [28]. Each deletion in E. coli cells was confirmed by PCR as described in [28]. For cell growth analysis, overnight cell cultures grown in Luria-Bertani (LB) medium were washed twice with minimal medium containing glucose (0.2%) before inoculated on minimal medium plates or in liquid minimal medium at 37°C with aeration (250 rpm). Cell growth was recorded by measuring the optical density of cell culture at 600 nm. When indicated, minimal medium was supplemented with the three branched-chain amino acids ...

Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins

“…In contrast, the suggested role of the A-type ISC assembly proteins as alternative Fe/S scaffold proteins that work in parallel to the Isu proteins seems unlikely in yeast. Rather, the Isa proteins and Iba57 form a specialized ISC subcomplex that operates downstream of the central components of the mitochondrial ISC assembly system and is specifically required for maturation of mitochondrial (27,32).…”

“…Escherichia coli harbors three canonical A-type proteins, IscA, SufA, and ErpA, and the non-canonical NfuA. Deletion of iscA is not associated with a clear phenotype, whereas that of ErpA or a double deletion of iscA and sufA results in strong growth defects (26,27). These phenotypes frequently aggravate under special growth conditions, such as iron limitation or oxidative stress, and are linked to the loss of function of crucial cellular Fe/S proteins (3, 21, 26 -33).…”

Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I

“…In this respect, we can envision huInd1 as a specialized biogenesis factor which donates Fe/S clusters to specific apoproteins at the final stage of Fe/S protein maturation. Another interesting group of proteins playing a similar specialized role is yeast Isa1, Isa2, and Iba57 and the bacterial counterparts, all of which are dedicated to the maturation of aconitase-type and radical S-adenosylmethionine Fe/S proteins (18,32,64). It is likely that future studies will reveal several more proteins apart from the core Fe/S cluster biogenesis components that perform specialized functions, e.g., in specifically delivering Fe/S clusters to apoproteins both inside and outside mitochondria.…”

Stress Response and Virulence Functions of the Acinetobacter baumannii NfuA Fe-S Scaffold Protein

“…All pathways involve a cysteine desulfurase and a U-type and/or A-type scaffold protein capable of assembling both [2Fe-2S] and [4Fe-4S] clusters (3). U-type scaffold proteins, such as NifU and IscU, preferentially bind [2Fe-2S] clusters while Atype scaffold proteins, such as SufA and IscA, can bind [2Fe-2S] monomeric clusters, which then mature to [4Fe-4S]-containing proteins, as it is in the case of aconitase (23,37). More recent studies led to the observation that E. coli (1) and A. vinelandii (4) produce an additional protein involved in [Fe-S] biogenesis that was named NfuA.…”