Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe–S cluster synthesis

2012 ◽  
Vol 448 (2) ◽  
pp. 171-187 ◽  
Author(s):  
Alok Pandey ◽  
Ramesh Golla ◽  
Heeyong Yoon ◽  
Andrew Dancis ◽  
Debkumar Pain
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Viability ◽  
Conformational Change ◽  
Active Site ◽  
Enzyme Activation ◽  
Accessory Proteins ◽  
Cysteine Desulfurase ◽  
Interacting Protein ◽  
Active Site Cysteine ◽  
Isolated Mitochondria

Cysteine desulfurases abstract sulfur from the substrate cysteine, generate a covalent persulfide on the active site cysteine of the enzyme, and then donate the persulfide sulfur to various recipients such as Fe–S clusters. In Saccharomyces cerevisiae, the Nfs1p protein is the only known cysteine desulfurase, and it forms a complex with Isd11p (Nfs1p·Isd11p). Both of these proteins are found primarily in mitochondria and both are essential for cell viability. In the present study we show, using the results of experiments with isolated mitochondria and purified proteins, that Isd11p is required for the cysteine desulfurase activity of Nfs1p. Whereas Nfs1p by itself was inactive, the Nfs1p·Isd11p complex formed persulfide and was active as a cysteine desulfurase. In the absence of Isd11p, Nfs1p was able to bind the substrate cysteine but failed to form a persulfide. Addition of Isd11p allowed Nfs1p with bound substrate to generate a covalent persulfide. We suggest that Isd11p induces an activating conformational change in Nfs1p to bring the bound substrate and the active site cysteine in proximity for persulfide formation. Thus mitochondrial Nfs1p is different from bacterial cysteine desulfurases that are active in the absence of accessory proteins. Isd11p may serve to regulate cysteine desulfurase activity in mitochondria.

scholarly journals Substitutions in an Active Site Loop ofEscherichia coliIscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesisin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 19551-19558 ◽  
Author(s):  
Charles T. Lauhon ◽  
Elizabeth Skovran ◽  
Hugo D. Urbina ◽  
Diana M. Downs ◽  
Larry E. Vickery
Keyword(s):  
Active Site ◽  
Cluster Formation ◽  
Wild Type ◽  
Cysteine Desulfurase ◽  
Mutant Proteins ◽  
Active Site Cysteine ◽  
Sulfur Transfer ◽  
Amino Acid Region

IscS catalyzes the fragmentation ofl-cysteine tol-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. InEscherichia coliIscS, the active site cysteine Cys328resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328identified additional residues important for IscS functionin vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesisin vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer.In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesisin vivowithout detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

scholarly journals Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

1999 ◽  
Vol 10 (5) ◽  
pp. 1367-1379 ◽  
Author(s):  
Isei Tanida ◽  
Noboru Mizushima ◽  
Miho Kiyooka ◽  
Mariko Ohsumi ◽  
Takashi Ueno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Cysteine Residue ◽  
Atp Binding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Significant Similarity ◽  
Active Site Cysteine ◽  
Thioester Bond ◽  
Two Hybrid ◽  
Novel Protein

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

scholarly journals Saccharomyces cerevisiae SMT4 Encodes an Evolutionarily Conserved Protease With a Role in Chromosome Condensation Regulation

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 95-107 ◽  
Author(s):  
Alexander V Strunnikov ◽  
L Aravind ◽  
Eugene V Koonin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Site Specific ◽  
Active Site Cysteine ◽  
Condensin Complex ◽  
Evolutionarily Conserved ◽  
Synthetically Lethal

Abstract In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo. The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption. The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion. We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.

Alkylation of Escherichia coli Thioredoxin by S-(2-Chloroethyl)glutathione and Identification of the Adduct on the Active Site Cysteine-32 by Mass Spectrometry

1995 ◽  
Vol 8 (7) ◽  
pp. 934-941 ◽  
Author(s):  
John C. L. Erve ◽  
Elisabeth Barofsky ◽  
Douglas F. Barofsky ◽  
Max L. Deinzer ◽  
Donald J. Reed
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Active Site ◽  
Active Site Cysteine

scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

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