TY - JOUR A1 - Zupok, Arkadiusz A1 - Górka, Michał Jakub A1 - Siemiatkowska, Beata A1 - Skirycz, Aleksandra A1 - Leimkühler, Silke T1 - Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli JF - Journal of bacteriology N2 - Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes. KW - Escherichia coli KW - FNR KW - iron regulation KW - iron-sulfur cluster KW - anaerobic respiration KW - molybdenum cofactor Y1 - 2019 U6 - https://doi.org/10.1128/JB.00382-19 SN - 0021-9193 SN - 1098-5530 VL - 201 IS - 17 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Mendel, Ralf R. A1 - Hercher, Thomas W. A1 - Zupok, Arkadiusz A1 - Hasnat, Muhammad Abrar A1 - Leimkühler, Silke T1 - The requirement of inorganic Fe-S clusters for the biosynthesis of the organometallic molybdenum cofactor JF - Inorganics : open access journal N2 - Iron-sulfur (Fe-S) clusters are essential protein cofactors. In enzymes, they are present either in the rhombic [2Fe-2S] or the cubic [4Fe-4S] form, where they are involved in catalysis and electron transfer and in the biosynthesis of metal-containing prosthetic groups like the molybdenum cofactor (Moco). Here, we give an overview of the assembly of Fe-S clusters in bacteria and humans and present their connection to the Moco biosynthesis pathway. In all organisms, Fe-S cluster assembly starts with the abstraction of sulfur froml-cysteine and its transfer to a scaffold protein. After formation, Fe-S clusters are transferred to carrier proteins that insert them into recipient apo-proteins. In eukaryotes like humans and plants, Fe-S cluster assembly takes place both in mitochondria and in the cytosol. Both Moco biosynthesis and Fe-S cluster assembly are highly conserved among all kingdoms of life. Moco is a tricyclic pterin compound with molybdenum coordinated through its unique dithiolene group. Moco biosynthesis begins in the mitochondria in a Fe-S cluster dependent step involving radical/S-adenosylmethionine (SAM) chemistry. An intermediate is transferred to the cytosol where the dithiolene group is formed, to which molybdenum is finally added. Further connections between Fe-S cluster assembly and Moco biosynthesis are discussed in detail. KW - Moco biosynthesis KW - Fe-S cluster assembly KW - l-cysteine desulfurase KW - ISC KW - SUF KW - NIF KW - iron KW - molybdenum KW - sulfur Y1 - 2020 U6 - https://doi.org/10.3390/inorganics8070043 SN - 2304-6740 VL - 8 IS - 7 PB - MDPI CY - Basel ER - TY - JOUR A1 - Hasnat, Muhammad Abrar A1 - Zupok, Arkadiusz A1 - Olas-Apelt, Justyna Jadwiga A1 - Müller-Röber, Bernd A1 - Leimkühler, Silke T1 - A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical S-adenosylmethionine protein MoaA for the synthesis of active molybdoenzymes JF - Journal of bacteriology N2 - Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression.
IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions. KW - iron-sulfur clusters KW - Moco biosynthesis KW - MoaA KW - A-type carrier protein KW - FNR KW - nitrate reductase KW - molybdenum cofactor Y1 - 2021 U6 - https://doi.org/10.1128/JB.00086-21 SN - 1098-5530 VL - 203 IS - 12 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Burschel, Sabrina A1 - Decovic, Doris Kreuzer A1 - Nuber, Franziska A1 - Stiller, Marie A1 - Hofmann, Maud A1 - Zupok, Arkadiusz A1 - Siemiatkowska, Beata A1 - Gorka, Michal Jakub A1 - Leimkühler, Silke A1 - Friedrich, Thorsten T1 - Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH BT - ubiquinone oxidoreductase (complex I) JF - Molecular microbiology N2 - The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b. Y1 - 2018 U6 - https://doi.org/10.1111/mmi.14137 SN - 0950-382X SN - 1365-2958 VL - 111 IS - 1 SP - 31 EP - 45 PB - Wiley CY - Hoboken ER -