Positively Charged Residues within the Iron–Sulfur Cluster Loop of E. coli MutY Participate in Damage Recognition and Removal

2000 ◽  
Vol 380 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Cindy Lou Chepanoske ◽  
Marie-Pierre Golinelli ◽  
Scott D. Williams ◽  
Sheila S. David
Keyword(s):  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Damage Recognition ◽  
Iron Sulfur ◽  
Charged Residues ◽  
Positively Charged

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

scholarly journals Zinc Toxicity and Iron-Sulfur Cluster Biogenesis inEscherichia coli

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Jianghui Li ◽  
Xiaojun Ren ◽  
Bingqian Fan ◽  
Zhaoyang Huang ◽  
Wu Wang ◽  
...  
Keyword(s):  
Binding Activity ◽  
Zinc Toxicity ◽  
Cluster Assembly ◽  
Intracellular Zinc ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Assembly Proteins

ABSTRACTWhile zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases inEscherichia coli. Here, we report that the intracellular zinc overload inE. colicells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene clusteriscSUA-hscBA-fdx-iscXinE. colicells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of theE. colimutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin inE. colicells.IMPORTANCEZinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload inEscherichia colicells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

scholarly journals The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e21992 ◽  
Author(s):  
Clara Iannuzzi ◽  
Salvatore Adinolfi ◽  
Barry D. Howes ◽  
Ricardo Garcia-Serres ◽  
Martin Clémancey ◽  
...  
Keyword(s):  
Scaffold Protein ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

scholarly journals In vivo evidence for the iron-binding activity of an iron–sulfur cluster assembly protein IscA in Escherichia coli

2010 ◽  
Vol 432 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Wu Wang ◽  
Hao Huang ◽  
Guoqiang Tan ◽  
Fan Si ◽  
Min Liu ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Binding Activity ◽  
Anaerobic Growth ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

IscA is a key member of the iron–sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron–sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron–sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron–sulfur clusters in E. coli cells under aerobic conditions.

scholarly journals Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Guoqiang Tan ◽  
Jing Yang ◽  
Tang Li ◽  
Jin Zhao ◽  
Shujuan Sun ◽  
...  
Keyword(s):  
Copper Toxicity ◽  
Anaerobic Conditions ◽  
Iron Sulfur Cluster ◽  
Aerobic Conditions ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins ◽  
Intracellular Copper

ABSTRACT While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they are under aerobic conditions. Under anaerobic conditions, E. coli cells accumulate excess intracellular copper, which specifically targets iron-sulfur proteins by blocking iron-sulfur cluster biogenesis. Since iron-sulfur proteins are involved in diverse and vital physiological processes, inhibition of iron-sulfur cluster biogenesis by copper disrupts multiple cellular functions and ultimately inhibits cell growth. The results from this study illustrate a new interplay between intracellular copper toxicity and iron-sulfur cluster biogenesis in bacterial cells under anaerobic conditions.

scholarly journals Elevated Expression of a Functional Suf Pathway in the E.coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein

2019 ◽  
Author(s):  
Elliot I. Corless ◽  
Erin L. Mettert ◽  
Patricia J. Kiley ◽  
Edwin Antony
Keyword(s):  
Large Scale ◽  
Biochemical Characterization ◽  
Protein A ◽  
Fold Increase ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Elevated Expression ◽  
Operon Expression

ABSTRACTStructural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is - inactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered ‘SufFeScient’ derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.IMPORTANCELarge quantities of recombinantly overexpressed iron-sulfur cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that increase Suf expression can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

scholarly journals Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Liliana S. O. Silva ◽  
Pedro M. Matias ◽  
Célia V. Romão ◽  
Lígia M. Saraiva
Keyword(s):  
Protein Interactions ◽  
Structural Basis ◽  
Single Mutation ◽  
Protein Protein Interactions ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Crucial Component

Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.

Complementary roles of SufA and IscA in the biogenesis of iron–sulfur clusters in Escherichia coli

2007 ◽  
Vol 409 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Jianxin Lu ◽  
Juanjuan Yang ◽  
Guoqiang Tan ◽  
Huangen Ding
Keyword(s):  
Cell Growth ◽  
Double Mutant ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Iron Binding Protein

Biogenesis of iron–sulfur clusters requires a concerted delivery of iron and sulfur to target proteins. It is now clear that sulfur in iron–sulfur clusters is derived from L-cysteine via cysteine desulfurases. However, the specific iron donor for the iron–sulfur cluster assembly still remains elusive. Previous studies showed that IscA, a member of the iron–sulfur cluster assembly machinery in Escherichia coli, is a novel iron-binding protein, and that the iron-bound IscA can provide iron for the iron–sulfur cluster assembly in a proposed scaffold IscU in vitro. However, genetic studies have indicated that IscA is not essential for the cell growth of E. coli. In the present paper, we report that SufA, an IscA paralogue in E. coli, may represent the redundant activity of IscA. Although deletion of IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA in E. coli results in a severe growth phenotype in minimal medium under aerobic growth conditions. Cell growth is restored when either IscA or SufA is re-introduced into the iscA−/sufA− double mutant, demonstrating further that either IscA or SufA is sufficient for their functions in vivo. Purified SufA, like IscA, is an iron-binding protein that can provide iron for the iron–sulfur cluster assembly in IscU in the presence of a thioredoxin reductase system which emulates the intracellular redox potential. Site-directed mutagenesis studies show that the SufA/IscA variants that lose the specific iron-binding activity fail to restore the cell growth of the iscA−/sufA− double mutant. The results suggest that SufA and IscA may constitute the redundant cellular activities to recruit intracellular iron and deliver iron for the iron–sulfur cluster assembly in E. coli.

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