Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis.

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone-mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein-mediated maturation of mitochondrial Fe/S proteins and the importance for human disease.

RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification.

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.

Bacterial cysteate dissimilatory pathway involves a racemase and d-cysteate sulfo-lyase.

The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H2S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H2S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments.

The structure of the SufS-SufE complex reveals interactions driving protected persulfide transfer in iron-sulfur cluster biogenesis.

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, pyridoxal 5'-phosphate-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using Escherichia coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ∼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces.

Persulfide Transfer to SufE Activates the Half-Sites Reactivity of the E. coli Cysteine Desulfurase SufS.

The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe-S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of ∼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation (k3) and a net rate constant for the downstream steps (k5). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s-1 and 0.10 ± 0.01 s-1 were determined for k3 and k5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k3 but induces a 10-fold enhancement of k5 to 1.1 ± 0.2 s-1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.

Selection of synthetic proteins to modulate the human frataxin function.

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus.

Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold and 73.9% of enzymatic recovery. The molecular mass of the purified MGL was approximately apparent at 46 KDa based on SDS-PAGE analysis. The enzymatic biochemical properties showed a maximum activity at pH 7 and exhibited plausible stability within pH range 5.0-7.5; meanwhile the highest catalytic activity of MGL was observed at 30-40 °C and the enzymatic stability was noted up to 40 °C. The enzyme molecule was significantly inhibited in the presence of Cu2+, Cd2+, Li2+, Mn2+, Hg2+, sodium azide, iodoacetate, and mercaptoethanol. Moreover, MGL displayed a maximum activity toward the following substrates, L-methionine < DL-methionine < Ethionine < Cysteine. Kinetic studies of MGL for L-methioninase showed catalytic activity at 20.608 mM and 12.34568 µM.min-1. Furthermore, MGL exhibited anticancer activity against cancerous cell lines, where IC50 were 243 ± 4.87 µg/ml (0.486 U/ml), and 726 ± 29.31 µg/ml (1.452 U/ml) against Hep-G2, and HCT116 respectively. In conclusion, A. fumigatus MGL had good catalytic properties along with significantly anticancer activity at low concentration which makes it a probably candidate to apply in the enzymotherapy field.

Mechanism of activation of the human cysteine desulfurase complex by frataxin.

The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to the incurable neurodegenerative disease Friedreich's ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondrial function. The structural and functional core of the Fe-S cluster assembly complex is a low-activity pyridoxal 5'-phosphate (PLP)-dependent cysteine desulfurase enzyme that consists of catalytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) subunits. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. Here, we develop a radiolabeling assay and use stopped-flow kinetics to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. Our results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. Our resulting architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.

Structure-Function Analysis of Interallelic Complementation in ROOTY Transheterozygotes.

Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in genes involved in glucosinolate biosynthesis, a metabolically related route that produces defense compounds, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic Arabidopsis (Arabidopsis thaliana) mutants for the essential glucosinolate biosynthetic gene ROOTY (RTY) that exhibit a range of phenotypic defects characteristic of enhanced auxin production. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 cyp79b3, wei2, and wei7 mutations and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY elucidated the relationships between structure and function in the RTY homo- and heterodimers, and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.

Integrated Succinylome and Metabolome Profiling Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of Aeromonas hydrophila.

Protein modification by lysine succinylation is a newly identified post-translational modification (PTM) of lysine residues and plays an important role in diverse physiological functions, although their associated biological characteristics are still largely unknown. Here, we investigated the effects of lysine succinylation on the physiological regulation within a well-known fish pathogen, Aeromonas hydrophila A high affinity purification method was used to enrich peptides with lysine succinylation in A. hydrophila ATCC 7966, and a total of 2,174 lysine succinylation sites were identified on 666 proteins using LC-MS/MS. Gene ontology analysis indicated that these succinylated proteins are involved in diverse metabolic pathways and biological processes, including translation, protein export, and central metabolic pathways. The modifications of several selected candidates were further validated by Western blotting. Using site-directed mutagenesis, we observed that the succinylation of lysines on S-ribosylhomocysteine lyase (LuxS) at the K23 and K30 sites positively regulate the production of the quorum sensing autoinducer AI-2, and that these PTMs ultimately alter its competitiveness with another pathogen, Vibrio alginolyticus Moreover, subsequent metabolomic analyses indicated that K30 succinylation on LuxS may suppress the activated methyl cycle (AMC) and that both the K23 and K30 sites are involved in amino acid metabolism. Taken together, the results from this study provide significant insights into the functions of lysine succinylation and its critical roles on LuxS in regulating the cellular physiology of A. hydrophila.

A Genetically Encoded Two-Dimensional Infrared Probe for Enzyme Active-Site Dynamics.

While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-L-tyrosine (N3Y) in the active-site of DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to FeIII , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

Relationship between activity and stability: Design and characterization of stable variants of human frataxin.

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS.

SufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli, this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically, this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five X-ray crystal structures of SufS including a new structure of SufS containing an inward-facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved ß-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data, identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.

Structure-Function Analysis Indicates that an Active-Site Water Molecule Participates in Dimethylsulfoniopropionate Cleavage by DddK.

The osmolyte dimethylsulfoniopropionate (DMSP) is produced in petagram quantities in marine environments and has important roles in global sulfur and carbon cycling. Many marine microorganisms catabolize DMSP via DMSP lyases, generating the climate-active gas dimethyl sulfide (DMS). DMS oxidation products participate in forming cloud condensation nuclei and, thus, may influence weather and climate. SAR11 bacteria are the most abundant marine heterotrophic bacteria; many of them contain the DMSP lyase DddK, and their dddK transcripts are relatively abundant in seawater. In a recently described catalytic mechanism for DddK, Tyr64 is predicted to act as the catalytic base initiating the ß-elimination reaction of DMSP. Tyr64 was proposed to be deprotonated by coordination to the metal cofactor or its neighboring His96. To further probe this mechanism, we purified and characterized the DddK protein from Pelagibacter ubique strain HTCC1062 and determined the crystal structures of wild-type DddK and its Y64A and Y122A mutants (bearing a change of Y to A at position 64 or 122, respectively), where the Y122A mutant is complexed with DMSP. The structural and mutational analyses largely support the catalytic role of Tyr64, but not the method of its deprotonation. Our data indicate that an active water molecule in the active site of DddK plays an important role in the deprotonation of Tyr64 and that this is far more likely than coordination to the metal or His96. Sequence alignment and phylogenetic analysis suggest that the proposed catalytic mechanism of DddK has universal significance. Our results provide new mechanistic insights into DddK and enrich our understanding of DMS generation by SAR11 bacteria.IMPORTANCE The climate-active gas dimethyl sulfide (DMS) plays an important role in global sulfur cycling and atmospheric chemistry. DMS is mainly produced through the bacterial cleavage of marine dimethylsulfoniopropionate (DMSP). When released into the atmosphere from the oceans, DMS can be photochemically oxidized into DMSO or sulfate aerosols, which form cloud condensation nuclei that influence the reflectivity of clouds and, thereby, global temperature. SAR11 bacteria are the most abundant marine heterotrophic bacteria, and many of them contain DMSP lyase DddK to cleave DMSP, generating DMS. In this study, based on structural analyses and mutational assays, we revealed the catalytic mechanism of DddK, which has universal significance in SAR11 bacteria. This study provides new insights into the catalytic mechanism of DddK, leading to a better understanding of how SAR11 bacteria generate DMS.

Application of l-methionine γ-lyase in chiral amino acid analysis.

Here, a conventional chiral amino acid analysis method using high-performance liquid chromatography was coupled with a sample pretreatment using l-methionine γ-lyase from Pseudomonas putida ICR 3460 for the selective analysis of l-methionine and l-tryptophan. The sample was analyzed after the degradation of l-methionine with l-methionine γ-lyase, as l-methionine coelutes with l-tryptophan under the standard chiral amino acid analytical conditions used for precolumn derivatization with o-phthalaldehyde and N-acetyl-l-cysteine. The l-tryptophan in the sample was then eluted as a clearly separated peak and analyzed further. Since the l-methionine γ-lyase did not act on l-tryptophan, we were able to calculate the l-methionine or l-tryptophan concentration based on the data obtained from 2 individual runs: the sample with and without l-methionine γ-lyase pretreatment. The concentration of l-tryptophan was calculated from the data obtained from the sample with l-methionine γ-lyase pretreatment, while the concentration of l-methionine was calculated using the following equation: l-methionine concentration = {the data from the sample without l-methionine γ-lyase pretreatment}-{the data from the sample with l-methionine γ-lyase pretreatment}. Model samples containing authentic amino acids and a fermented food sample were analyzed by our method, and the calculated concentrations of l-methionine and l-tryptophan were consistently in agreement with the theoretical values.

Recent progresses on synthesized LuxS inhibitors: A mini-review.

Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.

Functional analysis of iron-sulfur cluster biogenesis (SUF pathway) from Plasmodium vivax clinical isolates.

Iron-sulfur (Fe-S) clusters are critical metallo-cofactors required for cell function. Assembly of these cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. In Plasmodium, two pathways for these Fe-S cluster biogenesis have been reported; ISC pathway in the mitochondria and SUF pathway functional in the apicoplast. Amongst these, SUF pathway is reported essential for the apicoplast maintenance and parasite survival. Many of its components have been studied from P. falciparum and P. berghei in recent years, still few queries remain to be addressed; one of them being the assembly and transfer of Fe-S clusters. In this study, using P. vivax clinical isolates, we have shown the in vitro interaction of SUF pathway proteins SufS and SufE responsible for sulfur mobilization in the apicoplast. The sulfur mobilized by the SufSE complex assembles on the scaffold protein PvSufA along with iron provided by the external source. Here, we demonstrate in vitro transfer of these labile Fe-S clusters from the scaffold protein on to an apo-protein, PvIspG (a protein involved in penultimate step of Isoprenoids biosynthesis pathway) in order to provide an insight into the interaction of different components for the biosynthesis and transfer of Fe-S clusters. Our analysis indicate that inspite of the presence of variations in pathway proteins, the overall pathway remains well conserved in the clinical isolates when compared to that reported in lab strains.

Identification of a Heat-Inducible Element of Cysteine Desulfurase Gene Promoter in Lentinula edodes.

Volatile organosulfur compounds are the main components that contribute to the unique aroma of dried Lentinula edodes. They are mainly generated during the hot-air drying process, and cysteine desulfurase is the key enzyme in this process. Temperature may be an essential factor of volatile organosulfur compound production by influencing the expression of the cysteine desulfurase gene. In this study, the promoter sequence of the cysteine desulfurase gene (pCS) was cloned and analyzed using bioinformatics tools. A series of 5'deletion fragments and site-directed mutations of pCS were constructed to identify the element that responds to heat stress. Six heat shock transcription factor (HSTF) binding sites were predicted by SCPD (The Promoter Database of Saccharomyces cerevisiae) and three of the binding sites were predicted by Yeastract (Yeast Search for Transcriptional Regulators and Consensus Tracking) in pCS. The results indicated that pCS was able to drive the expression of the EGFP (Enhanced Green Fluorescent Protein) gene in L. edodes. Moreover, the fluorescence intensity increased after heat stress. The changes in fluorescence intensity of different 5'deletion fragments showed that the heat response region was located between -500 bp and -400 bp in pCS. The site-directed mutation analysis further showed that the heat-inducible element was between -490 bp and -500 bp (TTTCTAGAAT) in pCS. Our results provide molecular insight for studying the formation of volatile organosulfur compounds in dried L. edodes.