Sulfur Carrier Research Articles

Ubiquitin-like prokaryotic MoaD as a fusion tag for expression of heterologous proteins in Escherichia coli

BackgroundEukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.ResultsFusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.ConclusionsProkaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.

Evaluation of a highly reactive and sulfur resistant synthetic Fe-based oxygen carrier for CLC using gaseous fuels

Because of its low cost and environmental compatibility, Fe-based oxygen carriers are now being considered as an attractive option for chemical-looping combustion (CLC) applications. In this work, a synthetic Fe-based oxygen carrier made by impregnation using Al2O3 as support was evaluated with respect to gas combustion in a 500 Wth CLC continuous unit using CH4 as fuel during long-term operation. Furthermore, a study on the fate of sulfur during CH4 combustion was carried out with this oxygen carrier.During the experimental tests, it was observed that an increase in the oxygen carrier-to-fuel ratio (φ) produces an increase in the combustion efficiency, reaching full gas combustion at φ values 2 1.5. In addition, the presence of sulfur in the fuel gas did not affect to the combustion efficiency, independently of the amount of sulfur in the gas stream in the range of 700 to 2000 vppm H2S. However, the attrition of this oxygen carrier was higher than other materials used in CLC.Therefore, it can be concluded that the synthetic Fe-based oxygen carrier developed in this work was highly reactive and sulfur resistant and it can be considered as a suitable oxygen carrier for CLC with gaseous fuels that contain sulfur when the attrition behavior was improved.

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

ThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. In Escherichia coli and Salmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, in Bacillus subtilis and most species from the Firmicutes phylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis of B. subtilis thiI and the adjacent gene, nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments in B. subtilis indicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine. In vitro synthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain. In vivo complementation studies in E. coli IscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal that B. subtilis NifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

Posttranslational Modification of Cellular Proteins by a Ubiquitin-like Protein in Bacteria

Posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins plays important regulatory roles in eukaryotes. Although a homologous conjugation system has recently been reported in Archaea, there is no similar report in Bacteria. This report describes the identification of a ubiquitin-like conjugation system in the bacterium Thermus thermophilus. A series of in vivo analyses revealed that TtuB, a bacterial ubiquitin-like protein that functions as a sulfur carrier in tRNA thiouridine synthesis, was covalently attached to target proteins, most likely via its C-terminal glycine. The involvement of the ubiquitin-activating enzyme-like protein TtuC in conjugate formation and the attachments of TtuB to TtuC and TtuA, which are proteins required for tRNA thiouridine synthesis, were demonstrated. Mass spectrometry analysis revealed that lysine residues (Lys-137/Lys-226/Lys-229) of TtuA were covalently modified by the C-terminal carboxylate of TtuB. Intriguingly, a deletion mutant of a JAMM (JAB1/MPN/Mov34 metalloenzyme) ubiquitin isopeptidase homolog showed aberrant TtuB conjugates of TtuC and TtuA and an ∼50% decrease in thiouridine amounts in tRNA. These results would support the hypothesis that thiouridine synthesis is regulated by TtuB-conjugation.

The dual role of ubiquitin-like protein Urm1 as a protein modifier and sulfur carrier

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.

Urm1 couples sulfur transfer to ubiquitin-like protein function in oxidative stress

The posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins (collectively referred to as UBLs) has emerged as a major regulatory mechanism in eukaryotes. UBLs are characterized by a core β-grasp fold and an essential carboxy terminal glycine within a di-glycine motif (1). These features are also found in several prokaryotic sulfur carriers, suggestive of an evolutionary relationship to UBLs (2–5). A case in point is the UBL Urm1 (ubiquitin-related modifier 1). Urm1 is known to be conjugated to the peroxiredoxin Ahp1 (6), but its sequence and structure more closely resembles bacterial sulfur carrier proteins (7). In PNAS, work by Van der Veen et al. provides important insight into the mechanism of Urm1 conjugation that highlights its position as an ancient UBL in eukaryotes and solidifies its roles as a posttranslational protein modifier involved in oxidative stress response mechanisms (8).

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

Biosynthesis of Thiamin Pyrophosphate.

The biosynthesis of thiamin pyrophosphate (TPP) in prokaryotes, as represented by the Escherichia coli and the Bacillus subtilis pathways, is summarized in this review. The thiazole heterocycle is formed by the convergence of three separate pathways. First, the condensation of glyceraldehyde 3-phosphate and pyruvate, catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), gives 1-deoxy-D-xylulose 5-phosphate (DXP). Next, the sulfur carrier protein ThiS-COO- is converted to its carboxyterminal thiocarboxylate in reactions catalyzed by ThiF, ThiI, and NifS (ThiF and IscS in B. subtilis). Finally, tyrosine (glycine in B. subtilis) is converted to dehydroglycine by ThiH (ThiO in B. subtilis). Thiazole synthase (ThiG) catalyzes the complex condensation of ThiS-COSH, dehydroglycine, and DXP to give a thiazole tautomer, which is then aromatized to carboxythiazole phosphate by TenI (B. subtilis). Hydroxymethyl pyrimidine phosphate (HMP-P) is formed by a complicated rearrangement reaction of 5-aminoimidazole ribotide (AIR) catalyzed by ThiC. ThiD then generates hydroxymethyl pyrimidine pyrophosphate. The coupling of the two heterocycles and decarboxylation, catalyzed by thiamin phosphate synthase (ThiE), gives thiamin phosphate. A final phosphorylation, catalyzed by ThiL, completes the biosynthesis of TPP, the biologically active form of the cofactor. This review reviews the current status of mechanistic and structural studies on the enzymes involved in this pathway. The availability of multiple orthologs of the thiamin biosynthetic enzymes has also greatly facilitated structural studies, and most of the thiamin biosynthetic and salvage enzymes have now been structurally characterized.

CHEMICAL MODELING OF YOUNG STELLAR OBJECTS. I. METHOD AND BENCHMARKS

Upcoming facilities such as the Herschel Space Observatory or Atacama Large Millimeter Array will deliver a wealth of molecular line observations of young stellar objects (YSOs). Based on line fluxes, chemical abundances can then be estimated by radiative transfer calculations. To derive physical properties from abundances, the chemical network needs to be modeled and fitted to the observations. This modeling process is however computationally exceedingly demanding, particularly if in addition to density and temperature, far-UV (FUV) irradiation, X-rays, and multi-dimensional geometry have to be considered. We develop a fast tool, suitable for various applications of chemical modeling in YSOs. A grid of the chemical composition of the gas having a density, temperature, FUV irradiation and X-ray flux is pre-calculated as a function of time. A specific interpolation approach is developed to reduce the database to a feasible size. Published models of AFGL 2591 are used to verify the accuracy of the method. A second benchmark test is carried out for FUV sensitive molecules. The novel method for chemical modeling is more than 250,000 times faster than direct modeling and agrees within a mean factor of 1.35. The tool is distributed for public use. Main applications are (1) fitting physical parameters to observed molecular line fluxes and (2) deriving chemical abundances for two- and three-dimensional models. They will be presented in two future publications of this series. In the course of developing the method, the chemical evolution is explored: we find that X-ray chemistry in envelopes of YSOs can be reproduced by means of an enhanced cosmic-ray ionization rate with deviations less than 25%, having the observational consequence that molecular tracers for X-rays are hard to distinguish from cosmic-ray ionization tracers. We provide the detailed prescription to implement this total ionization rate approach in any chemical model. We further find that the abundance of CH+ in low-density gas with high ionization can be enhanced by the recombination of doubly ionized carbon (C++) and suggest a new value for the initial abundance of the main sulfur carrier in the hot core.

Solution structure of Urm1 from Trypanosoma brucei

Solution structure of Urm1 from <i>Trypanosoma brucei</i>

The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.

O-Phospho-l-serine and the Thiocarboxylated Sulfur Carrier Protein CysO-COSH Are Substrates for CysM, a Cysteine Synthase from Mycobacterium tuberculosis

The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.

Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM−CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 Å resolution. CysM (Rv1336) is a PLP-containing β-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 Å resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM−CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF−ThiS and MoeB−MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

The Sulfurtransferase Activity of Uba4 Presents a Link between Ubiquitin-like Protein Conjugation and Activation of Sulfur Carrier Proteins

Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.

Determination of sulfur in steels by radiochemical neutron activation analysis with liquid scintillation counting

A method has been developed for the determination of low-level sulfur in steels by radiochemical neutron activation analysis. During sample irradiation, 35S is produced by the 34S(n,γ)35S reaction. Irradiated steels are mixed with sulfur carrier and dissolved in HCl/HNO3. Sulfur is reduced to H2S by reaction with HI/H3PO2/HCl. The evolved H2S is absorbed in dilute NaOH, which is mixed with scintillation cocktail for the measurement of 35S by liquid scintillation counting. Sulfur carrier yield is determined by iodometric titration. Chlorine is also determined by RNAA in order to correct for 35S produced via the 35Cl(n,p)35S reaction. Sulfur has been determined at mass fractions as low as ≈5 mg/kg in ultra-high-purity iron using this method.

Biosynthesis of the Thioquinolobactin Siderophore: an Interesting Variation on Sulfur Transfer

The thioquinolobactin siderophore from Pseudomonas fluorescens ATCC 17400 utilizes a variation of the sulfur transfer chemistry found in thiamine and molydobterin biosynthesis. A JAMM motif protein cleaves the C-terminal amino acid residues following a diglycine moiety on a small sulfur carrier protein, and the modified C terminus is activated and sulfurylated, forming a thiocarboxylate.

Solution structure of Urm1 and its implications for the origin of protein modifiers

Protein modifiers are involved in diverse biological processes and regulate the activity or function of target proteins by covalently conjugating to them. Although ubiquitin and a number of ubiquitin-like protein modifiers (Ubls) in eukaryotes have been identified, no protein modifier has been found in prokaryotes; thus, their evolutionary origin remains a puzzle. To infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins, we solved the solution NMR structure of the Urm1 (ubiquitin-related modifier-1) protein from Saccharomyces cerevisiae. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique "molecular fossil" that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD (molybdopterin synthase small subunit) suggest that they may interact with partners in a similar manner, and similarities between Urm1-Uba4 and MoaD-MoeB establish an evolutionary link between ATP-dependent protein conjugation in eukaryotes and ATP-dependent cofactor sulfuration.

Elucidation and Structural Analysis of Conserved Pools for Genome-Scale Metabolic Reconstructions

In this article, we introduce metabolite concentration coupling analysis (MCCA) to study conservation relationships for metabolite concentrations in genome-scale metabolic networks. The analysis allows the global identification of subsets of metabolites whose concentrations are always coupled within common conserved pools. Also, the minimal conserved pool identification (MCPI) procedure is developed for elucidating conserved pools for targeted metabolites without computing the entire basis conservation relationships. The approaches are demonstrated on genome-scale metabolic reconstructions of Helicobacter pylori, Escherichia coli, and Saccharomyces cerevisiae. Despite significant differences in the size and complexity of the examined organism's models, we find that the concentrations of nearly all metabolites are coupled within a relatively small number of subsets. These correspond to the overall exchange of carbon molecules into and out of the networks, interconversion of energy and redox cofactors, and the transfer of nitrogen, sulfur, phosphate, coenzyme A, and acyl carrier protein moieties among metabolites. The presence of large conserved pools can be viewed as global biophysical barriers protecting cellular systems from stresses, maintaining coordinated interconversions between key metabolites, and providing an additional mode of global metabolic regulation. The developed approaches thus provide novel and versatile tools for elucidating coupling relationships between metabolite concentrations with implications in biotechnological and medical applications.

The Biosynthesis of the Thiazole Phosphate Moiety of ThiaminThe Sulfur Transfer Mediated by the Sulfur Carrier Protein ThiS

Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using 18O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.