• Journal of the Korean Magnetic Resonance Society
• /
• v.28 no.1
• /
• pp.1-5
• /
• 2024

IscU, the iron-sulfur (Fe-S) cluster scaffold protein, is an essential protein for biogenesis of Fe-S clusters. Previous studies showed that IscU manifests a metamorphic structural feature; at least two structural states, namely the structured state (S-state) and the disordered state (D-state), interconverting in a physiological condition, was observed. Moreover, subsequent studies demonstrated that the metamorphic flexibility of IscU is important for its Fe-S cluster assembly activity as well as for an efficient interaction with various partner proteins. Although solution nuclear magnetic resonance (NMR) spectroscopy has been a useful tool to investigate this protein, the detailed molecular mechanism that sustains the structural heterogeneity of IscU is still unclear. To tackle this issue, we applied a high-pressure NMR (HP-NMR) technique to the IscU variant, IscU(I8K), which shows an increased population of the S-state. We found that the equilibrium between the S- and D-state was significantly perturbed by pressure application, and the specific regions of IscU exhibited more sensitivity to pressure than the other regions. Our results provide novel insights to appreciate the dynamic behaviors of IscU and the related versatile functionality.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2006.05a
• /
• pp.69-71
• /
• 2006

• Journal of Microbiology and Biotechnology
• /
• v.32 no.9
• /
• pp.1110-1119
• /
• 2022

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe₂(SO₄)₃ for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also upregulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

• Journal of Photoscience
• /
• v.9 no.2
• /
• pp.388-390
• /
• 2002

Two ferredoxin (Fd) fractions, namely, Fd-A and Fd-B were isolated from Heliobacillus mobilis cells, and purified by ammonium sulfate fractionation, DEAE, gel-permeation and Phenyl-Superose column chromatographies under anaerobic conditions. Their absorption spectra were typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. Their N-terminal amino acid sequences were determined, which showed that both of them contain a [4Fe-4S] cluster binding motif. Fd-B was sensitive to oxygen, and itsA$_{385}$ value decreased by about 50% in 2 h at 4$^{\circ}C$ under aerobic conditions. In contrast, $A_{385}$ of Fd-A was essentially unchanged up to 24 h under the same conditions.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.4
• /
• pp.438-447
• /
• 2011

Deinococcus radiodurans is extremely resistant to various genotoxic conditions and chemicals. In this study, we characterized the effect of a sublethal concentration (100 ${\mu}M$) of cadmium (Cd) on D. radiodurans using a whole-genome DNA microarray. Time-course global gene expression profiling showed that 1,505 genes out of 3,116 total ORFs were differentially expressed more than 2-fold in response to Cd treatment for at least one timepoint. The majority of the upregulated genes are related to iron uptake, cysteine biosynthesis, protein disulfide stress, and various types of DNA repair systems. The enhanced upregulation of genes involved in cysteine biosynthesis and disulfide stress indicate that Cd has a high affinity for sulfur compounds. Provocation of iron deficiency and growth resumption of Cd-treated cells by iron supplementation also indicates that CdS forms in iron-sulfur-containing proteins such as the [Fe-S] cluster. Induction of base excision, mismatch, and recombinational repair systems indicates that various types of DNA damage, especially base excision, were enhanced by Cd. Exposure to sublethal Cd stress reduces the growth rate, and many of the downregulated genes are related to cell growth, including biosynthesis of cell membrane, translation, and transcription. The differential expression of 52 regulatory genes suggests a dynamic operation of complex regulatory networks by Cd-induced stress. These results demonstrate the effect of Cd exposure on D. radiodurans and how the related genes are expressed by this stress.

• Journal of Photoscience
• /
• v.3 no.2
• /
• pp.77-83
• /
• 1996

Photosensitization via the singlet oxygen ($^1O_2$) mechanism by the binuclear iron-sulfur center, denoted as [2Fe-2S], was investigated, using a highly purified ferredoxin (Fd) preparation from spinach leaves. Since the apoprotein of Fd contains a good number of amino acid residues that are readily reactive with $^1O_2$ and thus interfere with the detection of $^1O_2$ generated from [2Fe-2S], we attempted to deprive the $^1O_2$-sensitive residues of their $^1O_2$-scavenging capacity as much as possible by treating Fd with rose bengal plus 550 nm monochromatic light and thereby photooxidatively degrading these residues. The photochemically modified Fd was found to keep the structural integrity of its Fe-S group virtually unaffected by the treatment. By employing chemical trap method for measurement and examining the kinetic effects of azide and deuterium oxide on the reactions of $^1O_2$ with various trap compounds, we were able to demonstrate that [2Fe-2S] indeed acts as a photosensitizer via $^1O_2$. Further, the minimum quantum yield of $^1O_2$ production by [2Fe-2S] was estimated to be 0.0047.

• Genomics & Informatics
• /
• v.21 no.1
• /
• pp.5.1-5.13
• /
• 2023

Neisseria gonorrhoeae is a Gram-negative aerobic diplococcus bacterium that primarily causes sexually transmitted infections through direct human sexual contact. It is a major public health threat due to its impact on reproductive health, the widespread presence of antimicrobial resistance, and the lack of a vaccine. In this study, we used a bioinformatics approach and performed subtractive genomic methods to identify potential drug targets against the core proteome of N. gonorrhoeae (12 strains). In total, 12,300 protein sequences were retrieved, and paralogous proteins were removed using CD-HIT. The remaining sequences were analyzed for non-homology against the human proteome and gut microbiota, and screened for broad-spectrum analysis, druggability, and anti-target analysis. The proteins were also characterized for unique interactions between the host and pathogen through metabolic pathway analysis. Based on the subtractive genomic approach and subcellular localization, we identified one cytoplasmic protein, 2Fe-2S iron-sulfur cluster binding domain-containing protein (NGFG RS03485), as a potential drug target. This protein could be further exploited for drug development to create new medications and therapeutic agents for the treatment of N. gonorrhoeae infections.

• Journal of the Korean Magnetic Resonance Society
• /
• v.17 no.2
• /
• pp.105-110
• /
• 2013

HP1492 is a NifU-like protein of Helicobacter pylori (H. pylori) and plays a role as a scaffold which transfer Fe-S cluster to Fe-S proteins like Ferredoxin. To understand how to bind to iron ion or iron-sulfur cluster, HP1492 was expressed and purified in Escherichia coli (E. coli). From the NMR measurement, we could carry out the sequence specific backbone resonance assignment of HP1492. Approximately 91% of all resonances could be assigned unambiguously. By analyzing results of CSI and TALOS from NMR data, we could predict the secondary structure of HP1492, which consists of three ${\alpha}$-helices and three ${\beta}$-sheets. This study is an essential step towards the structural characterization of HP1492.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1520-1526
• /
• 2009

We have identified the iscR (PA3815) gene encoding an iron-sulfur cluster assembly regulator homolog as one of the genes required for peroxide resistance in Pseudomonas aeruginosa PA14. Here, we present the phenotypic characterization of an iscR deletion mutant in terms of KatA expression, stress responses, and virulence. The iscR null mutant exhibited reduced KatA activity at the posttranslational level, hypersensitivity to hydrogen peroxide, and virulence-attenuation in Drosophila melanogaster and mouse peritonitis models. These phenotypes were fully restored by multicopy-based expression of katA. These results suggest that the requirement of IscR in P. aeruginosa is related to the proper activity of KatA, which is crucial for peroxide resistance and full virulence of this bacterium.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.8
• /
• pp.1142-1148
• /
• 2020

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.