Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains.

We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.

Transcatheter aortic valve implantation after aortic valve neocuspidization using autologous pericardium: a case report.

BACKGROUND: Aortic valve neocuspidization (AVNeo), a novel surgical procedure used in the treatment of aortic valve diseases, including aortic stenosis (AS), involves the replacement of three aortic valve cusps by glutaraldehyde-treated autologous pericardium. Although reoperation risk is low, no case report on the deterioration of the AVNeo has yet been published. CASE SUMMARY: An 80-year-old woman who underwent AVNeo for severe degenerative tricuspid AS 6 years previously complained of shortness of breath. Echocardiographic assessment revealed the reconstructed aortic valve leaflet was elongated, thickened, and marginally calcified resulting in recurrent severe AS. Transcatheter aortic valve implantation using balloon-expandable transcatheter heart valve was successfully performed. DISCUSSION: To our knowledge, this is the first case report regarding the structural deterioration of the AVNeo resulting in restenosis 6 years after the first surgery. Transcatheter aortic valve implantation is possibly a suitable approach for post-procedural recurrence after AVNeo to avoid redo open-heart surgery which would be of prohibitive risk especially in an elderly population.

Identification of IscU residues critical for de novo iron-sulfur cluster assembly.

IscU is a central component of the ISC machinery and serves as a scaffold for the de novo assembly of iron-sulfur (Fe-S) clusters prior to their delivery to target apo-Fe-S proteins. However, the molecular mechanism is not yet fully understood. In this study, we have conducted mutational analysis of E. coli IscU using the recently developed genetic complementation system of a mutant that can survive without Fe-S clusters. The Fe-S cluster ligands (C37, C63, H105, C106) and the proximal D39 and K103 residues are essential for in vivo function of IscU and could not be substituted with any other amino acids. Furthermore, we found that substitution of Y3, a strictly conserved residue among IscU homologs, abolished in vivo functions. Surprisingly, a second-site suppressor mutation in IscS (A349V) reverted the defect caused by IscU Y3 substitutions. Biochemical analysis revealed that IscU Y3 was crucial for functional interaction with IscS and sulfur transfer between the two proteins. Our findings suggest that the critical role of IscU Y3 is linked to the conformational dynamics of the flexible loop of IscS, which is required for the ingenious sulfur transfer to IscU.

Generation of hydrogen sulfide from sulfur assimilation in Escherichia coli.

Many organisms produce endogenous hydrogen sulfide (H2S) as a by-product of protein, peptide, or L-cysteine degradation. Recent reports concerning mammalian cells have demonstrated that H2S acts as a signaling molecule playing important roles in various biological processes. In contrast to mammals, bacterial H2S signaling remains unclear. In this work, we demonstrate that Escherichia coli generates H2S through the assimilation of inorganic sulfur, without L-cysteine degradation. Comparison of phenotypes and genomes between laboratory E. coli K-12 strains revealed a major contribution of CRP (a protein that controls the expression of numerous genes involved in glycolysis) to H2S generation. We found that H2S was produced by cells growing in a synthetic minimal medium containing thiosulfate as a sole inorganic sulfur source, but not in a medium only containing sulfate. Furthermore, E. coli generated H2S in a CRP-dependent manner as a response to glucose starvation. These results indicate that CRP plays a key role in the generation of H2S coupled to thiosulfate assimilation, whose molecular mechanisms remains to be elucidated. Here, we propose a potential biological role of the H2S as a signaling mediator for a cross-talk between carbon and sulfur metabolism in E. coli.

Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli.

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (L-cysteine (Cys), L-histidine, and L-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Distinct roles for U-type proteins in iron-sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon.

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.

Mapping the key residues of SufB and SufD essential for biosynthesis of iron-sulfur clusters.

Biogenesis of iron-sulfur (Fe-S) clusters is an indispensable process in living cells. In Escherichia coli, the SUF biosynthetic system consists of six proteins among which SufB, SufC and SufD form the SufBCD complex, which serves as a scaffold for the assembly of nascent Fe-S cluster. Despite recent progress in biochemical and structural studies, little is known about the specific regions providing the scaffold. Here we present a systematic mutational analysis of SufB and SufD and map their critical residues in two distinct regions. One region is located on the N-terminal side of the ß-helix core domain of SufB, where biochemical studies revealed that Cys254 of SufB (SufBC254) is essential for sulfur-transfer from SufE. Another functional region resides at an interface between SufB and SufD, where three residues (SufBC405, SufBE434, and SufDH360) appear to comprise the site for de novo cluster formation. Furthermore, we demonstrate a plausible tunnel in the ß-helix core domain of SufB through which the sulfur species may be transferred from SufBC254 to SufBC405. In contrast, a canonical Fe-S cluster binding motif (CxxCxxxC) of SufB is dispensable. These findings provide new insights into the mechanism of Fe-S cluster assembly by the SufBCD complex.

Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli.

Sulfate (SO42-) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (S2O32-), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO32-) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2-) â†’ L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.

Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron-sulfur clusters.

Biological assembly of iron-sulfur (Fe-S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA-hscBA-fdx-iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe-S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe-S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe-S cluster-containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron-transfer Fe-S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe-S scaffold protein responsible for the de novo Fe-S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe-S cluster biosynthesis.

Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis.

ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.

Immunolocalization of 8-5' and 8-8' linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies.

Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5' or 8-8' linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA-BSA and negatively reacted with pAHA-BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA-BSA conjugates containing 8-O-4' linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4', 8-8', or 5-5' linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4', 8-5', or 5-5' linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5' and 8-8' linked structure of lignin in the cell walls.