scholarly journals Iron-Sulfur Cluster Assembly

2004 ◽  
Vol 279 (19) ◽  
pp. 19705-19711 ◽  
Author(s):  
Patricia C. Dos Santos ◽  
Archer D. Smith ◽  
Jeverson Frazzon ◽  
Valerie L. Cash ◽  
Michael K. Johnson ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Cluster Formation ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Molecular Scaffold ◽  
Terminal Domain ◽  
Related Family ◽  
Fe Protein

The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. Thesein vivoexperiments were supported byin vitro[Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.

scholarly journals Iron–sulfur cluster biosynthesis

2008 ◽  
Vol 36 (6) ◽  
pp. 1112-1119 ◽  
Author(s):  
Sibali Bandyopadhyay ◽  
Kala Chandramouli ◽  
Michael K. Johnson
Keyword(s):  
Scaffold Proteins ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Eukaryotic Organisms ◽  
Almost All ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

scholarly journals Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

2013 ◽  
Vol 288 (20) ◽  
pp. 14200-14211 ◽  
Author(s):  
Sylvain Boutigny ◽  
Avneesh Saini ◽  
Edward E. K. Baidoo ◽  
Natasha Yeung ◽  
Jay D. Keasling ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Carrier Protein ◽  
Cluster Assembly ◽  
Carrier Proteins ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Initial Cluster ◽  
Initial Assembly

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-l-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

scholarly journals IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

2013 ◽  
Vol 288 (23) ◽  
pp. 16680-16689 ◽  
Author(s):  
Mineaki Seki ◽  
Yukiko Takeda ◽  
Kazuhiro Iwai ◽  
Kiyoji Tanaka
Keyword(s):  
Cluster Assembly ◽  
Core Complex ◽  
Core Component ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
The Core ◽  
Iron Sulfur ◽  
External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

scholarly journals Mitochondrial Iba57p Is Required for Fe/S Cluster Formation on Aconitase and Activation of Radical SAM Enzymes

2007 ◽  
Vol 28 (5) ◽  
pp. 1851-1861 ◽  
Author(s):  
Cristy Gelling ◽  
Ian W. Dawes ◽  
Nadine Richhardt ◽  
Roland Lill ◽  
Ulrich Mühlenhoff
Keyword(s):  
Cluster Formation ◽  
Cluster Assembly ◽  
Radical Sam ◽  
Radical Sam Enzymes ◽  
Functional Studies ◽  
A Genome ◽  
Assembly Factor ◽  
S Proteins

ABSTRACT A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57Δ mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57Δ growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

A Sensitive Method for Assay of Mixed-Function Oxygenase (p-450 complex) in Cell Culture

1988 ◽  
Vol 15 (3) ◽  
pp. 219-223
Author(s):  
Jørgen Clausen ◽  
Søren Achim Nielsen
Keyword(s):  
Human Lymphocytes ◽  
Cellular Systems ◽  
Metabolic Effects ◽  
Assay Method ◽  
Related Family ◽  
Oxygenase Activity ◽  
Metabolic Transformation ◽  
Mixed Function Oxygenase

The mixed-function oxygenase system involved in the metabolism of drugs and xenobiotics has been extensively studied in various animal species and in various organs (1). It is now apparent that in humans the p-450 complex is one representative of a related family, expressed by 13 c-DNA genes showing approximately 36% similarity between the different subfamilies (2). In order to compare the in vivo and in vitro metabolic effects of drugs and xenobiotics, the induction capabilities of the mixed-function oxygenase must be known. The most sensitive non-isotopic assay system for determination of mixed-function oxygenase activity is the method of Nebert & Gelboin (3,4), which is based on the metabolic transformation of benzo-(a)-pyrene to its fluorescent hydroxyl derivatives (5). However, the levels of the mixed-function oxygenase enzymes in different cellular systems show great variations, with the highest activities in liver cells. Therefore, in order to use human lymphocytes and other cellular systems with low mixed-function oxygenase activities, the assay method for determining oxygenase activity must have the highest possible sensitivity. The present communication is devoted to a study aimed at increasing the sensitivity of Nebert & Gelboin's methods for assay of mixed-function oxygenase subfamilies using benzo-(a)-pyrene as a substrate.

scholarly journals Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

2021 ◽  
Vol 22 (4) ◽  
pp. 1598
Author(s):  
Amber L. Hendricks ◽  
Christine Wachnowsky ◽  
Brian Fries ◽  
Insiya Fidai ◽  
James A. Cowan
Keyword(s):  
Cluster Assembly ◽  
Paramagnetic Resonance ◽  
Iron Sulfur Cluster ◽  
Sulfur Transfer ◽  
Disease States ◽  
Isolation And Characterization ◽  
Iron Sulfur ◽  
Natural Iron ◽  
Lipoyl Synthase

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.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

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

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
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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