Faculty Opinions recommendation of Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

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MRE11 Is Crucial for Malaria Parasite Transmission and Its Absence Affects Expression of Interconnected Networks of Key Genes Essential for Life

Cells ◽

10.3390/cells9122590 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2590

Author(s):

David S. Guttery ◽

Abhinay Ramaprasad ◽

David J. P. Ferguson ◽

Mohammad Zeeshan ◽

Rajan Pandey ◽

...

Keyword(s):

Genome Stability ◽

Malaria Parasite ◽

Mosquito Vector ◽

Parasite Transmission ◽

Biological Processes ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Parasite Plasmodium ◽

Damage Response

The meiotic recombination 11 protein (MRE11) plays a key role in DNA damage response and maintenance of genome stability. However, little is known about its function during development of the malaria parasite Plasmodium. Here, we present a functional, ultrastructural and transcriptomic analysis of Plasmodium parasites lacking MRE11 during its life cycle in both mammalian and mosquito vector hosts. Genetic disruption of Plasmodium berghei mre11 (PbMRE11) results in significant retardation of oocyst development in the mosquito midgut associated with cytoplasmic and nuclear degeneration, along with concomitant ablation of sporogony and subsequent parasite transmission. Further, absence of PbMRE11 results in significant transcriptional downregulation of genes involved in key interconnected biological processes that are fundamental to all eukaryotic life including ribonucleoprotein biogenesis, spliceosome function and iron–sulfur cluster assembly. Overall, our study provides a comprehensive functional analysis of MRE11′s role in Plasmodium development during the mosquito stages and offers a potential target for therapeutic intervention during malaria parasite transmission.

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Multiple Sense and Antisense Promoters Contribute to the Regulated Expression of the isc-suf Operon for Iron-Sulfur Cluster Assembly in Rhodobacter

Microorganisms ◽

10.3390/microorganisms7120671 ◽

2019 ◽

Vol 7 (12) ◽

pp. 671 ◽

Cited By ~ 1

Author(s):

Xin Nie ◽

Bernhard Remes ◽

Gabriele Klug

Keyword(s):

Rhodobacter Sphaeroides ◽

Photosynthetic Electron Transport ◽

Regulatory Proteins ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Iron Sulfur Clusters ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Photosynthetic Complexes ◽

The One

A multitude of biological functions relies on iron-sulfur clusters. The formation of photosynthetic complexes goes along with an additional demand for iron-sulfur clusters for bacteriochlorophyll synthesis and photosynthetic electron transport. However, photooxidative stress leads to the destruction of iron-sulfur clusters, and the released iron promotes the formation of further reactive oxygen species. A balanced regulation of iron-sulfur cluster synthesis is required to guarantee the supply of this cofactor, on the one hand, but also to limit stress, on the other hand. The phototrophic alpha-proteobacterium Rhodobacter sphaeroides harbors a large operon for iron-sulfur cluster assembly comprising the iscRS and suf genes. IscR (iron-sulfur cluster regulator) is an iron-dependent regulator of isc-suf genes and other genes with a role in iron metabolism. We applied reporter gene fusions to identify promoters of the isc-suf operon and studied their activity alone or in combination under different conditions. Gel-retardation assays showed the binding of regulatory proteins to individual promoters. Our results demonstrated that several promoters in a sense and antisense direction influenced isc-suf expression and the binding of the IscR, Irr, and OxyR regulatory proteins to individual promoters. These findings demonstrated a complex regulatory network of several promoters and regulatory proteins that helped to adjust iron-sulfur cluster assembly to changing conditions in Rhodobacter sphaeroides.

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Regulation of the HscA ATPase Reaction Cycle by the Co-chaperone HscB and the Iron-Sulfur Cluster Assembly Protein IscU

Journal of Biological Chemistry ◽

10.1074/jbc.m410117200 ◽

2004 ◽

Vol 279 (52) ◽

pp. 53924-53931 ◽

Cited By ~ 59

Author(s):

Jonathan J. Silberg ◽

Tim L. Tapley ◽

Kevin G. Hoff ◽

Larry E. Vickery

Keyword(s):

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Reaction Cycle ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Atpase Reaction

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Interchangeability and Distinct Properties of Bacterial Fe-S Cluster Assembly Systems: Functional Replacement of the isc and suf Operons in Escherichia coli with the nifSU-Like Operon from Helicobacter pylori

The Journal of Biochemistry ◽

10.1093/jb/mvh104 ◽

2004 ◽

Vol 136 (2) ◽

pp. 199-209 ◽

Cited By ~ 83

Author(s):

U. Tokumoto

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Assembly Systems ◽

Cluster Assembly ◽

Functional Replacement

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Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

Journal of Biological Chemistry ◽

10.1074/jbc.m113.467001 ◽

2013 ◽

Vol 288 (20) ◽

pp. 13951-13959 ◽

Cited By ~ 9

Author(s):

Yan Zhang ◽

Xiuxiang An ◽

JoAnne Stubbe ◽

Mingxia Huang

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribonucleotide Reductase ◽

Large Subunit ◽

Small Subunit ◽

Cluster Assembly ◽

E Coli ◽

Viability Assay ◽

Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

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