ABSTRACT
Iron acquisition by siderophores is crucial for survival and virulence of many microorganisms. Here, we investigated the binding of the exogenous siderophore ferric enterobactin and the synthetic siderophore mimic ferric mecam by the triscatecholate binding protein FeuA from Bacillus subtilis at the atomic level. The structural complexes provide molecular insights into the capture mechanism of FeuA for exogenous and synthetic siderophores. The protein-ligand complexes show an exclusive acceptance of Λ-stereoconfigured substrates. Ligand-induced cross-bridging of the complexes was not observed, revealing a different thermodynamic behavior especially of the ferric mecam substrate, which was previously shown to dimerize with the enterobactin binding protein CeuE. The nearly identical overall domain movement of FeuA upon binding of ferric enterobactin or ferric mecam compared with endogenously derived ferric bacillibactin implies the importance of the conserved domain rearrangement for recognition by the transmembrane permease FeuBC, for which the conserved FeuA residues E90 and E221 were proved to be essential.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Siderophores/metabolism , Bacillus subtilis/chemistry , Binding Sites , Crystallography, X-Ray , Enterobactin/metabolism , Ferric Compounds/metabolism , Models, Molecular , Protein BindingABSTRACT
Frataxin homologues are important iron chaperones in eukarya and prokarya. Using a native proteomics approach we were able to identify the structural frataxin homologue Fra (formerly YdhG) of Bacillus subtilis and to quantify its native iron-binding stoichiometry. Using recombinant proteins we could show in vitro that Fra is able to transfer iron onto the B. subtilis SUF system for iron-sulfur cluster biosynthesis. In a four-constituents reconstitution system (including SufU, SufS, Fra and CitB) we observed a Fra-dependent formation of a [4 Fe-4 S] cluster on SufU that could be efficiently transferred onto the target apo-aconitase (CitB). A Δfra deletion mutant showed a severe growth phenotype associated with a broadly disturbed iron homeostasis; this indicates that Fra is a central component of intracellular iron channeling in B. subtilis.
Subject(s)
Bacillus subtilis/metabolism , Iron-Binding Proteins/metabolism , Iron/metabolism , Bacillus subtilis/genetics , Iron-Binding Proteins/chemistry , Iron-Binding Proteins/genetics , Mass Spectrometry , Molecular Chaperones/metabolism , Proteomics , FrataxinABSTRACT
Iron-sulfur cluster biosynthesis in Gram-positive bacteria is mediated by the SUF system. The transfer of sulfide from the cysteine desulfurase SufS to the scaffold protein SufU is one of the first steps within the assembly process. In this study, we analyzed the interaction between Bacillus subtilis SufS and its scaffold SufU. The activity of SufS represents a Ping-Pong mechanism leading to successive sulfur loading of the conserved cysteine residues in SufU. Cysteine 41 of SufU is shown to be essential for receiving sulfide from SufS, while cysteines 66 and 128 are needed for SufS/SufU interaction. In conclusion, we present the first step-by-step model for loading of the essential scaffold component SufU by its sulfur donor SufS.
Subject(s)
Bacillus subtilis/metabolism , Biocatalysis , Carbon-Sulfur Lyases/chemistry , Carbon-Sulfur Lyases/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Sulfur/chemistry , Amino Acid Substitution , Bacillus subtilis/enzymology , Catalytic Domain , Cysteine/metabolism , Gram-Positive Bacteria/enzymology , Gram-Positive Bacteria/metabolism , Iron/chemistry , Kinetics , Models, Molecular , Protein Interaction Domains and Motifs , Recombinant Proteins , Sulfur/metabolismSubject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Benzamides/metabolism , Chromatography, Affinity , Siderophores/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Benzamides/chemistry , Ligands , Mutation , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium's genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to approximately 1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.
