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Keywords:

  • persulfide;
  • sulfurtransferase;
  • desulfurase;
  • cystine lyase;
  • prokaryotic sulfur metabolism;
  • ubiquitin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Sulfur is a functionally important element of living matter. Incorporation into biomolecules occurs by two basic strategies. Sulfide is added to an activated acceptor in the biosynthesis of cysteine, from which methionine, coenzyme A and a number of biologically important thiols can be constructed. By contrast, the biosyntheses of iron sulfur clusters, cofactors such as thiamin, molybdopterin, biotin and lipoic acid, and the thio modification of tRNA require an activated sulfur species termed persulfidic sulfur (R-S-SH) instead of sulfide. Persulfidic sulfur is produced enzymatically with the IscS protein, the SufS protein and rhodanese being the most prominent biocatalysts. This review gives an overview of sulfur incorporation into biomolecules in prokaryotes with a special emphasis on the properties and the enzymatic generation of persulfidic sulfur as well as its use in biosynthetic pathways.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Sulfur adds considerable functionality to a wide variety of biomolecules because of its unique properties: its chemical bonds are made as well as broken easily and sulfur equally well serves as an electrophile (e.g. in disulfides) and as a nucleophile (e.g. as thiol) (Beinert, 2000a, b). For incorporation into biomolecules, sulfur must be reduced and/or activated, and sulfate or polysulfides are substrates for reductases that are widespread in nature. With sulfide as the final product of reduction, incorporation into cysteine is possible and this amino acid serves as a central building block for many sulfur compounds; these compounds are discussed only briefly in this review. Another category of sulfur compounds is biosynthesized using an activated form of sulfur, which is termed ‘sulfane sulfur’ or (more specifically) ‘persulfidic sulfur’ (R-S-SH). The focus of this review is on the properties, the enzymatic generation and the use of this ‘activated sulfur’. Much of our knowledge regarding these subjects is derived from microbiological studies or biochemical work on microbiologically important processes such as nitrogen fixation and vitamin synthesis.

Sulfur-containing biomolecules

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Sulfide incorporation into an activated acceptor

The use of sulfide for biosynthetic purposes requires the activation of the acceptor compound serine as an ester in bacteria as well as in plants (Fig. 1). Evidently, sulfide, although toxic, can be tolerated by living cells in concentrations that allow cysteine synthesis. Reported Km-values of O-acetylserine sulfhydrylases for sulfide are in the range 6 μM–5.2 mM (http://www.brenda.uni-koeln.de) but many reports give Km-values of around 0.5 mM. Direct estimates of cellular sulfide concentrations have only rarely been reported and fall in the range 20–160 μM (Schmidt & Jäger, 1992; Wang, 2002; Theissen et al., 2003). It is interesting to note that cysteine as a free amino acid is also rather toxic to cells and maintained at a low steady-state level of 100–200 μM (Park & Imlay, 2003).

image

Figure 1.  Biosynthesis of cysteine and overview of sulfur-containing biomolecules based on cysteine. THF, tetrahydrofolate.

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With cysteine as the central building block, several important metabolites (coenzyme A, methionine, S-adenosylmethionine; Fig. 1) can be constructed. The penicillin sulfur is also derived from cysteine, and various specialized thiol compounds contain cysteine either in peptidic structures (glutathione, trypanothione) or linked to a disaccharide (mycothiol) (Hand & Honek, 2005). More elaborate cyclical thiol compounds such as ergothioneine and ovothiol A are also biosynthesized at the expense of cysteine. They are specialized to inactivate compounds generated during oxidative stress conditions such as HO·, peroxynitrite or H2O2 (for a review see Hand & Honek, 2005). Derivatives of glutathione have been reported for certain bacteria: glutathione amide was found in Chromatium as a representative of anaerobic sulfur bacteria (Bartsch et al., 1996); and γ-glutamylcysteine and thiosulfate were identified as the major low-molecular-weight thiols in Halobacteria (Newton & Javor, 1985).

Other specialized thiols termed CoM and CoB are crucial for methane production performed by methanogenic Archaea. Their biosyntheses involve the addition of sulfide to an aldehyde as an activated acceptor species (Graham & White, 2002). The source of sulfide in vivo seems to be cysteine after some transformation by a biosynthetic enzyme but characterization of the enzyme reaction(s) is still in progress. If an IscS-like enzyme is involved, CoM and CoB would need to be regrouped to the sulfur compounds requiring ‘activated sulfur’ for their synthesis (see below). In the context of biological sulfide provision, it is interesting to note that the mode of cysteine biosynthesis in many methanogenic Archaea is different from the pathway depicted in Fig. 1. It was shown for Methanococcus maripaludis that the sole route for cysteine biosynthesis is tRNA-dependent. In a first step tRNACys becomes acylated with O-phosphoserine; in a second step O-phosphoserine-tRNACys is transformed into Cys-tRNACys using a sulfur donor that has yet to be identified (Sauerwald et al., 2005).

Incorporation of ‘activated sulfur’

As early as the 1980s sulfane sulfur, i.e. sulfur with a formal oxidation number of zero, was suggested to be the biologically relevant active sulfur species. Early evidence centered on sulfurtransferase enzymes such as rhodanese and mercaptopyruvate sulfurtransferase (Cerletti, 1986; Toohey, 1989). With the characterization of the NifS protein of Azotobacter (Zheng et al., 1993) and proteins related to this prototypic cysteine desulfurase (see below), the concept of sulfane sulfur or persulfidic sulfur as an activated sulfur source in living organisms has gained much support. Proteins that generate and use persulfidic sulfur have been identified in a number of basic metabolic processes especially in the biosyntheses of sulfur-containing vitamins and cofactors (Fig. 2). The main part of this review will deal in detail with the properties of persulfidic sulfur, its enzymatic generation and its use in specific biochemical pathways. Most of the biochemical data were originally obtained from bacterial systems, especially Escherichia coli, Azotobacter vinelandii, Salmonella enterica serovar Typhimurium, Bacillus subtilis or B. sphaericus, Synechocystis and Erwinia amylophora. The concept emerging from these studies is of general relevance as much of the prokaryotic biochemistry described in this review also applies to eukaryotes.

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Figure 2.  Sulfur compounds relying on persulfidic sulfur (R-S-SH) as sulfur source.

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Chemical properties of persulfidic sulfur

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Low-molecular-weight persulfides (R-S-SH) are generally quite labile, decomposing into thiol (R-SH) and elemental sulfur (S) under most conditions (Eq. 1).

  • image(1)

Direct experimental proof for persulfide products of enzymatic reactions could only be obtained by chemical stabilization via alkylation (Flavin, 1962) or cyclization (Lang & Kessler, 1999). Both derivatization reactions are favored by the increased acidity of perthiols where the S–H bond is weakened by ≈90 kJM−1 as compared with thiols (Everett & Wardman, 1995). Persulfide groups display a broad absorbance band at 335–350 nm. Because the absorption coefficient is very low (460 M−1 cm−1, Cavallini et al., 1970; 310 M−1 cm−1, Wood, 1987), the general method to detect and quantify sulfane sulfur is cyanolysis whereby thiocyanate (SCN) is formed (Eq. 2; Wood, 1987). This compound can easily be determined colorimetrically when complexed to ferric iron.

  • image(2)

In addition to persulfides, other sulfane sulfur compounds such as thiosulfate (SO3S), thiosulfonate ion (RSO3S), polysulfides (RSSnSR) or polythionates (SO3SnSO3) also react and can be discriminated by their reactivity towards cyanide (Wood, 1987).

Although low-molecular-weight persulfides are very labile as previously stated, the persulfide derivative of glutathione as well as its amide seem to exist transiently with a half-life sufficient to have some biological role.

Careful analysis of enzymatic sulfur-oxidation by Acidithiobacillus and Acidiphilium spp. identified glutathione persulfide (GSSH) as the substrate of glutathione-dependent sulfur dioxygenase, which could be shown to catalyse Eq. 3 (Rohwerder & Sand, 2003):

  • image(3)

The glutathione persulfide was formed in situ in the reaction mixtures either from glutathione and elemental sulfur (GSH+S0[RIGHTWARDS ARROW]GSSH) or from glutathione disulfide and hydrogen sulfide (GSSG+H2S[RIGHTWARDS ARROW]GSSH+GSH). Combining the enzymological data on the dioxygenase with other data in the literature, a general scheme for elemental sulfur oxidation in acidophilic sulfur bacteria was proposed that suggests mobilization of extracellular elemental sulfur (S8) as cysteinyl persulfide of an as yet unidentified outer membrane protein. This protein-based cysteinyl persulfide should serve as the in vivo substrate of the periplasmic sulfur dioxygenase (Rohwerder & Sand, 2003) either directly or after sulfur transfer to a periplasmic acceptor. Glutathione persulfide therefore seems to be at least an in vitro substrate for sulfur dioxygenase.

Even more significant with respect to a biological role, the persulfide of glutathione amide was shown to exist intracellularly in Chromatium when grown photoautotrophically on sulfide. Glutathione amide bears an amide group at the glycyl moiety of glutathione and is especially resistant to autoxidation (Bartsch et al., 1996). Cell extraction in the presence of monobromobimane, which reacts with thiol groups to give fluorescent derivatives, revealed that nearly all of the glutathione amide was in the persulfide state when Chromatium strains were cultured photoautotrophically (Bartsch et al., 1996). As Chromatium and anaerobic sulfur bacteria in general use hydrogen sulfide as a reductant for photosynthesis and produce sulfur, the persulfide of glutathione amide may play a role in the transfer of S0 to or from sulfur globules in these cells.

It is interesting to note that a prominent low-molecular-weight thiol of Halobacteria again represents a sulfane sulfur compound although a more stable one: thiosulfate was found in these cells besides γ-glutamylcysteine which is synthesized instead of glutathione in Halobacteria. It was shown that thiosulfate stems from oxidation of cysteine (Newton & Javor, 1985).

In summary it can be concluded that free low-molecular-weight persulfides are unstable but can occur transiently in cells, especially when elemental sulfur, which has poor solubility, has to be handled in biochemical processes.

Enzymes generating persulfidic sulfur

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Much of the aforementioned instability of persulfides can be overcome when these species are produced in the sheltered environment of an enzyme active site, and proteins generating and binding persulfidic sulfur have been characterized in great detail by classical enzymological investigations as well as by analysis of their three-dimensional structure. Results of these various approaches are presented in the subsections below each headed by the name of the protein to be discussed.

NifS

Characterization of the NifS protein as a cysteine desulfurase that generates an enzyme-based cysteinyl persulfide as a crucial intermediate emerged from the work of Dennis Dean and coworkers dedicated to the maturation of nitrogenase (Zheng et al., 1993). The NifS protein was suggested to provide the sulfur needed to construct the complex metalloclusters of nitrogenase in vivo. A second protein termed NifU serves as an acceptor for NifS–sulfur and as an assembly platform for cluster precursors (Smith et al., 2005). For further details concerning biological FeS cluster formation see section FeS clusters. Although the NifS/NifU system seems to be specialized in the maturation of nitrogenase it is not restricted to nitrogen-fixing organisms and may even have a general role in the maturation of FeS proteins in Helicobacter pylori (Olson et al., 2000) or Entamoeba histolytica (Ali et al., 2004).

The enzymology of the NifS protein was elucidated using the enzyme of A. vinelandii (Zheng et al., 1993, 1994), which therefore serves as a prototype for NifS as well as IscS proteins (see section IscS). A pyridoxal phosphate cofactor attached to the lysine in an His-Lys-X-X-X-Pro-X-Gly-X-Gly motif (Lys 202 in A. vinelandii NifS; X is any amino acid) is a crucial feature of these proteins. Additionally crucial is a conserved cysteinyl residue which serves as the persulfide site and is located in the C-terminal part of the sequence (Cys 325 in A. vinelandii NifS). NifS binds and transforms the cysteine substrate in a manner usual for pyridoxal phosphate-containing enzymes up to the stage of the central quinonoid intermediate (Fig. 3, left). The cysteine sulfur of this intermediate is then attacked by NifS cysteinyl residue 325 to produce the NifS persulfide and the alanine enamine bound to pyridoxal phosphate (Fig. 3, right). As already noted in the original publication on the mechanism (Zheng et al., 1994) this reaction is unique and was not previously known for biological systems. Perhaps the property of the pyridoxal phosphate-stabilized alanine carbanion as a poor leaving group makes NifS a modest catalyst with a specific activity of about 90 mU mg−1 (Zheng et al., 1993). It is important to note that activity values of NifS proteins in vitro are significantly affected by the presence of thiols in two ways. First, in the absence of a physiological sulfur acceptor, the thiols present in the reaction mixture (excess substrate cysteine plus usually dithiothreitol) have to regenerate the NifS persulfide site reductively. Second, thiol groups have the capacity to add to the pyridoxal phosphate cofactor instead of the amino group of the amino acid substrate, which influences the kinetic behavior. This was analysed in depth for the Slr0387 NifS/IscS homolog of Synechocystis (Behshad et al., 2004) and may explain why conflicting kinetic data on the NifS class of enzymes can be found in the literature.

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Figure 3.  Cleavage of the C – S bond of cysteine bound to the pyridoxal phosphate cofactor of NifS.

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The structures of the NifS protein of Thermotoga maritima (PDB accession number 1EG5) and the E. coli IscS protein (PDB accession number 1P3W) have been solved to 2.00 and 2.10 Å resolution, respectively (Kaiser et al., 2000; Cupp-Vickery et al., 2003). Both enzymes show the same architecture. They are homodimers and belong to the α-family of pyridoxal phosphate-dependent enzymes. Each monomer is subdivided into a large domain harboring the pyridoxal phosphate cofactor and a small domain where the critical cysteinyl residue is located in the middle of a loop. Unfortunately in both structures this loop is either completely (Kaiser et al., 2000) or partially (Cupp-Vickery et al., 2003) disordered and the molecular events depicted in Fig. 3 cannot be integrated into a structural frame. However, it should be noted that in the IscS crystals the partially ordered loop is directed away from the active site pocket, resulting in an estimated distance of greater than 17 Å between the crucial cysteinyl residue and the pyridoxal phosphate cofactor (Cupp-Vickery et al., 2003). A large conformational change should therefore accompany the attack of the NifS/IscS-cysteinyl residue on the substrate cysteine bound to pyridoxal phosphate (Fig. 3). Perhaps the crystallized conformation depicts the state of NifS/IscS passing the activated sulfur to a downstream acceptor.

IscS

The IscS protein has much in common biochemically with NifS although it fulfills more general roles in the cell. This became evident when IscS was identified in A. vinelandii and it was found that in contrast to NifS its gene could not be inactivated (Zheng et al., 1998). The iscS gene of A. vinelandii is located at the 5′ end of an operon, which also contains iscU, iscA, hscB, hscA and fdx; this type of operon is widespread in nature (Zheng et al., 1998) and crucial for general iron sulfur cluster (isc) biosynthesis in many organisms. IscU is the scaffold protein interacting with IscS analogous to the interaction of NifS with NifU although there are several differences when comparing the two systems in more detail (Johnson et al., 2005; see section FeS clusters). IscA is suggested to be either an alternative scaffold protein or to have some role in iron delivery to IscU (Ding et al., 2005), HscB and HscA are specialized chaperones, and Fdx is a 2Fe–2S ferredoxin with an as yet poorly defined redox role (Johnson et al., 2005). The interplay of these various components in FeS cluster biosynthesis has been most thoroughly studied in E. coli and yeast in which a prokaryotic type of machinery is contained in the mitochondrion and delivers essential components of the FeS assembly process to the other cell compartments (Lill & Mühlenhoff, 2005). Interestingly, this situation is also true for the mitochondrial remnant organelles (mitosomes) in Giardia, which contain IscS and IscU proteins, as revealed by various immunodetection methods (Tovar et al., 2003).

From work with E. coli it became evident that IscS has the role of a central sulfur-providing enzyme in that organism given that, in addition to the FeS clusters, the thionucleosides 4-thiouridine and 5-methylaminomethyl-2-thiouridine (Lauhon & Kambampati, 2000; Lauhon, 2002) as well as thiamin (Lauhon & Kambampati, 2000) also receive their sulfur from IscS. Indeed, other sulfur-containing molecules may be biosynthesized at the expense of IscS-activated sulfur although via more indirect pathways (see below).

The central importance of IscS in E. coli has motivated innumerable searches for sequence homologs in other organisms and often iscS-type genes can be identified by the encoded IscS protein signature motifs and the neighborhood to other isc-type components. However, it was found that IscS-related sequences fall into two groups, groups I and II (Mihara et al., 1997). NifS and IscS as described above are the founding members of group I, which is the more homogeneous group. Group II proteins differ from group I in four sequence regions each encompassing from 15 to 30 amino acid residues (Fig. 4; Mihara et al., 1997). Additionally, group II sequences are far more divergent than group I sequences (Tachezy et al., 2001) and probably display a greater biochemical versatility. For example in E. coli, two group II proteins exist, which were originally named CsdB (Mihara et al., 1999) or SufS (Patzer & Hantke, 1999), and CSD (Mihara et al., 1997), and described as selenocysteine lyase (Mihara et al., 1999) and cysteine sulfinate desulfinase (Mihara et al., 1997), respectively (see Table 1). However, both proteins seem to contribute to minor pathways of FeS cluster biosynthesis in E. coli, most probably by mobilizing sulfur from l-cysteine as substrate (Takahashi & Tokumoto, 2002; Loiseau et al., 2005). The suf system is of more general importance in other organisms (Takahashi & Tokumoto, 2002; Fontecave et al., 2005) and SufS is therefore described in more detail in the next section.

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Figure 4.  Schematic alignment of IscS group I and group II sequences. Regions in which the sequences of the two groups differ markedly from each other are shown in black; gaps in the alignment are bridged. The positions of crucial catalytic residues (His, Lys, Cys) are indicated by arrows.

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Table 1.   Designations and classification of cysteine desulfurases
NameGroupSynonyms
  • *

    Slr, Synechocystis open reading frame longer than 100 codons, reading direction right.

NifSI
IscSISlr0387*
SufSIICsdB (selenocysteine lyase); Slr0077*
CSD (cysteine sulfinate desulfinase)IICsdA

SufS

The SufS protein is the best biochemically characterized NifS/IscS group II protein, with those from E. coli (Ollagnier-de-Choudens et al., 2003; Outten et al., 2003), Erwinia chrysanthemi (Loiseau et al., 2003) and Synechocystis (Kessler, 2004; Tirupati et al., 2004) being the most extensively studied. Although cysteine desulfuration is expected to proceed by a mechanism analogous to that of NifS/IscS group I (see Fig. 3), the reaction is even more sluggish with specific activities in the range 8–25 mU mg−1 (Loiseau et al., 2003; Ollagnier-de-Choudens et al., 2003; Outten et al., 2003). This was attributed to the extremely ineffective cleavage of the C–S bond of the cysteine substrate. The cleavage step was found to be rate-limiting, at variance with the group I NifS/IscS proteins, and a rate constant of 0.02 s−1 was determined using Synechocystis SufS (Tirupati et al., 2004). Besides the NifS/IscS group I desulfurase activity, Synechocystis SufS was additionally found to possess cystine lyase activity (Kessler, 2004), which is also suited to produce persulfidic sulfur (see section Cystinelyase).

The sufS gene is part of the suf (sulfur utilization) operon, which comprises sufABCDSE in its complete form. However, sufBC seem to be the core components of the suf system given the representation of the various suf genes in sequenced microbial genomes (Takahashi & Tokumoto, 2002; Fontecave et al., 2005).

It was investigated whether SufS cysteine desulfurase activity would be enhanced by the presence of other Suf components; a significant stimulation (eight- to 47-fold) by SufE was found for the E. coli (Ollagnier-de-Choudens et al., 2003; Outten et al., 2003) and the E. chrysanthemi (Loiseau et al., 2003) enzymes. SufS and SufE have some tendency to form a complex (Loiseau et al., 2003) and this stimulation may be caused by a sulfur donor–acceptor relation of SufS to SufE involving one specific cysteinyl residue of each of the two proteins. Although crystal structures for SufS (Fujii et al., 2000; Lima, 2002) as well as SufE (Goldsmith-Fischman et al., 2004) are available, the molecular events leading to the activation of the desulfuration reaction and the transfer of persulfidic sulfur are not understood at present. The covalent catalytic cysteinyl residue of SufS, which is the putative sulfur donor site, is too far from the pyridoxal phosphate-bound cysteine to reflect the state of attack on the C–S bond, and the critical sulfur acceptor cysteinyl residue of SufE is buried inside the protein so that a direct transfer to this location can hardly be envisaged. Possibly the proteins exist in an alternative conformation in the SufS–SufE complex for which no structural data are yet available. Nevertheless, SufS should be thought of as a two-component enzyme that activates sulfur from cysteine to produce an SufE-based protein–persulfide product. It should be noted that the SufS desulfurase activity can be stimulated even further when the SufBCD complex is present in addition to SufE (Outten et al., 2003). From this observation, interaction of all these Suf proteins can be postulated (see Fig. 11).

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Figure 11.  Schematic and simplified representation of FeS cluster assembly machineries. Grey boxes indicate the putative sites of action of the ferredoxin Fdx and the chaperones HscA,B (see text).

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The importance of the suf system was initially underestimated as most of the work on IscS proteins of group I as well as group II had been done in E. coli, for which inactivation of the suf operon has no consequences under standard conditions. Nevertheless, suf mutations in E. coli proved to be lethal in an isc background and suf overexpression restores the growth phenotype of E. coli isc mutants (Takahashi & Tokumoto, 2002). It was further shown by microarray profiling that suf transcription is upregulated as part of the E. coli response to hydrogen peroxide (Zheng et al., 2001), which identified the importance of the Suf proteins during oxidative stress conditions. Another physiological challenge which activates Suf production is iron starvation (Outten et al., 2004). Transcriptional control of the suf operon in E. coli uses regulators of gene expression such as OxyR and IHF (integration host factor) during oxidative stress and the metalloregulatory protein Fur for induction by shortage of iron (Patzer & Hantke, 1999; Lee et al., 2004; Outten et al., 2004). It seems certain for E. coli and probably for all organisms harboring the suf system that Suf proteins play important roles in sulfur metabolism during stress. Furthermore, the suf operon is even essential for some species. Proven cases are Synechocystis (Tirupati et al., 2004), B. subtilis (Tirupati et al., 2004) and Mycobacterium smegmatis (Huet et al., 2005) but this list will certainly grow in the near future.

Rhodanese and rhodanese-like proteins

Rhodanese and rhodanese-like proteins generate enzyme-based persulfides, as do NifS, IscS and SufS. Thiosulfate is generally used as substrate for rhodaneses in in vitro assays, where cyanide is also included as a sulfur acceptor to regenerate the covalent catalytic cysteinyl residue (Eqn 4a and b):

  • image((4a))
  • image((4b))

In addition to cyanide, other thiophilic acceptor compounds are also acceptable; for instance, lipoate as well as thioredoxin have been shown to interact specifically with certain rhodaneses (Cianci et al., 2000; Ray et al., 2000). The physiological role and the physiological substrates of rhodaneses have long been debated and various roles have been suggested, with detoxification and FeS cluster assembly being the most prominent (Cerletti, 1986). More recently additional roles in thiamin, molybdopterin and thionucleoside biosynthesis have emerged, in which rhodanese-like protein modules are found in some of the proteins involved (see below). The difficulties in establishing the in vivo functions of rhodaneses lie in the redundancy of rhodanese modules and rhodanese activities. This seems to exclude the shaping of a phenotype upon inactivation of a rhodanese gene with the exception of a rhodanese-like gene of Saccharopolyspora erythraea, disruption of which resulted in cysteine auxotrophy in this Streptomycete (Donadio et al., 1990). The formation of thiosulfate is probably affected, which combines with O-acetyl serine in the Streptomyces pathway of cysteine biosynthesis.

A typical feature of the rhodanese superfamily is a modular structure of its various members (Bordo & Bork, 2002). The prototypic enzyme, bovine liver rhodanese, consists of an N-terminal inactive rhodanese module (catalytic cysteinyl residue replaced) and a C-terminal catalytic module, each encompassing about 120 amino acid residues. This domain organization is shared by mercaptopyruvate sulfurtransferase (see section Mercaptopyruvate sulfurtransferase) and is also typical for many rhodanese sequences distributed in all kingdoms. However, a new rhodanese-like protein has most recently been found in Halanaerobium congolense and other thiosulfate-reducing anaerobic bacteria and that harbors two potential catalytic sites and seems to be involved in thiosulfate reduction (Ravot et al., 2005).

Besides the two-domain rhodaneses, single-domain versions are known, with the E. coli GlpE protein as the prototype (Ray et al., 2000). For GlpE the double displacement mechanism of Eq. 4 is also valid. The glpE gene is a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli but its physiological function and possible association with the metabolism of glycerol phosphate remains to be established. Any implication for GlpE in the sulfur metabolism of E. coli is lacking (Ray et al., 2000). Another single-domain rhodanese of E. coli, PspE, is a periplasmic protein (Adams et al., 2002). Its gene belongs to the psp (phage-shock protein) operon, which is induced upon infection with filamentous phage and other stress conditions. Again it remains unclear how the sulfurtransferase activity of PspE might contribute to the physiological role of the Psp proteins, which is related to the maintenance of the membrane potential under stress conditions as shown for PspA (Kleerebezem et al., 1996).

Rhodanese modules may also be involved in processes besides sulfur transfer. An interesting example is the rhodanese homology domain of the E. coli Ybb protein, which is responsible for the exchange of sulfur for selenium in 2-thiouridine in vivo. Selenophosphate is used as source for selenium in the reaction but details of the mechanism remain unresolved (Wolfe et al., 2004). Nevertheless these data illustrate that rhodanese-like proteins are also players at the borderline between sulfur and selenium metabolism.

Mercaptopyruvate sulfurtransferase

Mercaptopyruvate sulfurtransferase catalyzes the reaction described in Eq. 5 which is similar to that for rhodanese. Mercaptopyruvate is transformed to pyruvate instead of thiosulfate to sulfite. Cyanide serves as sulfur acceptor:

  • image(5)

Mercaptopyruvate sulfurtransferases and rhodaneses are related in sequence and structure but mercaptopyruvate sulfurtransferases are only poorly characterized with regard to their enzymatic properties. Early kinetic investigations using the enzyme from bovine kidney indicated that a single displacement mechanism applies that may even lead to the release of free sulfur as the primary product (Jarabak & Westley, 1980). This sulfur then reacts either with thiols or with cyanide to yield persulfides or thiocyanate. Although an enzyme persulfide is not an intermediate of this reaction pathway a conserved cysteinyl residue is catalytically essential for mercaptopyruvate sulfurtransferase.

Recently a mercaptopyruvate sulfurtransferase of E. coli, SseA, was crystallized and a mechanism was proposed reconciling its kinetic behavior with structural data (Spallarossa et al., 2004). It was suggested that a disulfide intermediate involving substrate and active-site cysteine would be formed, which should serve as a donor compound of sulfane sulfur to an acceptor such as thiols or cyanide after isomerization to a thiosulfoxide (Fig. 5). Notably, a thiol acceptor would yield a diffusible persulfide product as an activated sulfur species. The isomerization reaction depicted in Fig. 5 is of general relevance for the labilization of sulfur atoms; however, thiosulfoxide formation has to be favored either by chemical properties of the disulfide (Toohey, 1989) or by interaction with active-site residues as postulated for mercaptopyruvate sulfurtransferase (Spallarossa et al., 2004).

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Figure 5.  Disulfide – thiosulfoxide isomerization reaction suggested to occur in the active site of the Escherichia coli mercaptopyruvate sulfurtransferase SseA.

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The physiological role of mercaptopyruvate sulfurtransferases is even less clear than that of rhodaneses. Suggestions include involvement in cysteine and methionine metabolism, detoxification, tRNA sulfuration or FeS cluster biosynthesis, but experimental evidence for each is still awaited. The E. coli mercaptopyruvate sulfurtransferase gene sseA was cloned as one of two genes that increase serine sensitivity of E. coli but it is unclear how this effect correlates with the mercaptopyruvate sulfurtransferase activity of SseA. Because the mercaptopyruvate sulfurtransferase sequences are clearly distinguished from the related rhodanese sequences with a specific active-site loop motif as a distinguishing feature (Bordo & Bork, 2002), mercaptopyruvate sulfurtransferases should fulfill nonoverlapping roles in sulfur metabolism that have yet to be discovered.

Cystine lyase

The cystine lyase reaction represents a pyridoxal phosphate-based β-elimination by which l-cysteine persulfide plus aminoacrylate are produced from l-cystine. Aminoacrylate is subsequently hydrolysed to yield pyruvate and ammonium ion as final products (Eq. 6):

  • image

This activity has been long established as a side-activity of cystathionase from rat liver (Cavallini et al., 1960), γ-cystathionase from Neurospora (Flavin, 1962) or β-cystathionase from E. coli (Delavier-Klutchko & Flavin, 1965). The β-cystathionase was purified later (among three other proteins) from E. coli extracts based on its ability to assist in the restoration of the FeS cluster of dihydroxyacid dehydratase in vitro (Flint et al., 1996). Despite their general ability to produce a cysteine persulfide product via the cystine lyase reaction, the metabolically relevant role of cystathionases lies in the interconversion of cysteine and methionine via the so-called transsulfuration pathways. The transsulfuration enzymes γ-cystathionase, β-cystathionase and cystathionine γ-synthase share extensive sequence homology (Steegborn et al., 1999) and are easily distiguished from the NifS sequence family from either group I or group II. However, the search for proteins involved in cyanobacterial FeS cluster biosynthesis using an in vitro assay for apo- to holoferredoxin conversion identified a unique cystine lyase related to NifS/IscS group II proteins based on significant overall sequence similarity (Leibrecht & Kessler, 1997; Lang & Kessler, 1999). This cystine lyase, named C-DES, lacks the critical covalent-catalytic cysteinyl residue of NifS-like proteins in its sequence (Lang & Kessler, 1999) and is therefore unable to generate a protein-based persulfide and alanine. Cystine is by far the preferred substrate for β-elimination (Eq. 6) instead of cysteine (Lang & Kessler, 1999), which can be rationalized on the basis of X-ray structural data (Clausen et al., 2000; Kaiser et al., 2003; Fig. 6). Both carboxylate groups of the substrate or of the products interact with arginine residues. Additionally, a hydrogen bond between the terminal sulfur of the cysteine persulfide product and His114-ND1 anchors the persulfide group to C-DES at a hydrophobic site that is nevertheless accessible from the surface of the dimeric enzyme (Clausen et al., 2000). However, an acceptor for the persulfidic sulfur has yet to be identified. Interestingly, all active site residues of C-DES (Fig. 6) have equivalents in the structure of the Synechocystis SufS protein (Tirupati et al., 2004) for which a cystine lyase activity has been reported in addition to the NifS-like activity (Kessler, 2004). Under standard growth conditions, SufS is the only essential nifS-like gene of Synechocystis. However, upon growth in dim light c-des mutants of Synechocystis show growth defects that are exacerbated by oxidative stress (D. Kessler, unpublished data). Because the c-des gene is well conserved across various cyanobacterial genomes it may be a specialized tool to provide persulfidic sulfur under the at least partially oxidizing conditions of oxygenic photosynthesis.

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Figure 6.  Active-site residues of the cystine lyase C-DES and interaction with the products of cystine cleavage (dashed lines).

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Cystine lyase activity is also associated with some other proteins of microbiological interest, even though its significance is not completely understood: cysteine conjugate β-lyases from various bacteria that are generally believed to be involved in the bioactivation of S-conjugates of xenobiotics (Vamvakas et al., 1988) can use cystine as substrate. The repressor of the maltose regulon of E. coli, MalY, displays both cystine lyase and cystathionase activities, with cystine being the favored substrate (Zdych et al., 1995). The biological role of the β-C-S lyase activity still remains to be established as enzymatic activity is not required for the repressor function (Zdych et al., 1995) and the way in which MalY interferes with mal gene expression in E. coli is only poorly understood. Cystalysin from the oral pathogen Treponema denticola prefers cysteine as substrate for β-elimination, which generates hydrogen sulfide; however, cystine is also cleaved efficiently and cysteine persulfide may have the role of a harmful reactive substance in this case (Krupka et al., 2000). Another toxin that probably exerts a cytotoxic effect through β-cleavage of cystine is the osteotoxin of Bordetella avium. This protein exclusively accepts S-substituted cysteine derivatives, with cystine being an efficient substrate (Gentry-Weeks et al., 1993).

Cysteine desulfidase

Several Archaebacteria surprisingly do not contain any homolog to the nifS/iscS/sufS genes (Takahashi & Tokumoto, 2002) but do contain FeS clusters in high abundance. Considering the concept of persulfidic sulfur generated from cyst(e)ine as precursor to cluster sulfide, activity of a different type could generate this sulfur species in Archaea (or another substrate might be used). Recently a 4Fe–4S enzyme termed l-cysteine desulfidase was isolated from Methanocaldococcus jannaschii that was proposed to generate cluster-bound sulfide from cysteine as substrate (Tchong et al., 2005; Fig. 7). It was suggested that this sulfide could be accepted by a disulfide contained in a receiver protein that would result in one thiol group plus one persulfide group. The persulfidic sulfur could then be passed to downstream acceptors as in the nif/isc systems (Tchong et al., 2005). Whether this concept will turn out to be valid awaits the identification of further protein components of the archaeal system as well as their functional characterization.

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Figure 7.  Proposed generation of cluster-bound sulfide by l-cysteine desulfidase.

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Specific biosynthetic pathways using ‘activated sulfur’

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Biosynthetic pathways that directly or indirectly rely on persulfidic sulfur were briefly introduced above (Fig. 2). They are explained in more depth in the following sections. It will become evident that persulfide generation generally takes place at the entrance to these pathways. Downstream steps reveal an interesting relation of thiamin and molybdenum cofactor biosynthesis with ubiquitin-dependent protein degradation. Biotin and lipoic acid biosyntheses require the activation of nonactivated C–H bonds besides activated sulfur. This difficult step is performed by members of the ‘Adomet radical’ or ‘radical SAM’ enzyme family, which are 4Fe–4S proteins. The synthesis of biotin and lipoic acid therefore depends on FeS cluster biosynthesis, which is itself a process requiring activated sulfur. A final subchapter is devoted to the biosyntheses of the various thionucleosides during which biochemical reaction modules of the other pathways discussed again show up.

Thiamin

Thiamin pyrophosphate is essential for all living organisms and performs a key role as the cofactor of enzymes involved in metabolism of carbohydrates and biosyntheses of branched chain amino acids. Thiamin biosynthesis in prokaryotes has been studied in considerable detail and has been lucidly reviewed (Begley et al., 1999). Two variant pathways exist and are exemplified by the situation in E. coli (Taylor et al., 1998; Xi et al., 2001) and S. enterica serovar Typhimurium (Webb et al., 1997; Skovran & Downs, 2000) on the one hand, and in B. subtilis (Park et al., 2003; Settembre et al., 2004) on the other hand. Only the steps of sulfur activation for introduction into the thiazole ring are discussed here (Fig. 8).

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Figure 8.  Generation of ThiS thiocarboxylate as sulfur donor for thiazole formation in Escherichia coli.

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The crucial sulfur-containing intermediate is the ThiS protein thiocarboxylate, which was elucidated from the E. coli system (Taylor et al., 1998). Alternatively, the acyldisulfide of ThiS thiocarboxylate and ThiF (another protein involved in thiamin biosynthesis) could be used in E. coli (Xi et al., 2001) but this intermediate could not be detected in the B. subtilis system (Park et al., 2003). ThiS thiocarboxylate is generated in a two-step process reminiscent of the coupling of ubiquitin to the ubiquitin-activating enzyme E1. In fact, the 7.3-kDa protein ThiS shares the C-terminal Gly–Gly sequence with ubiquitin whereas ThiF shares high sequence similarity with ubiquitin-activating enzyme. It is consistent with the mass spectroscopic analyses of ThiS, isolated from certain E. coli mutant strains, that ThiS is first adenylated by ThiF/ATP and then the activated sulfur is introduced by the action of IscS (Taylor et al., 1998; Fig. 8). In E. coli, Salmonella and Haemophilus an additional protein, ThiI, is involved that contains a rhodanese-like module, suggesting a persulfide carrier function (Webb et al., 1997; Palenchar et al., 2000; Fig. 9). Interestingly, several ThiF proteins also contain a rhodanese domain at their C-terminus (Bordo & Bork, 2002). Although obligate in E. coli, a ThiI homolog is lacking in B. subtilis.

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Figure 9.  Mechanistic suggestion for IscS and ThiI action during ThiS – ThiF acyldisulfide formation in Escherichia coli.

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A mechanism for IscS action has been suggested for the E. coli system (Xi et al., 2001; Fig. 9). However, this ends up in the disulfide intermediate, which could not be found in the Bacillus system. A simple reduction step may yield the ThiS thiocarboxylate plus IscS from the ThiS–IscS acyldisulfide in this case.

How the sulfur contained in the ThiS thiocarboxylate becomes incorporated into the thiazole again differs between E. coli and Bacillus. In E. coli, 1-deoxy-d-xylulose-5-phosphate plus tyrosine and the proteins ThiG and ThiH are involved; by contrast, in Bacillus, glycine and ThiO instead of tyrosine and ThiH supply the thiazole synthase ThiG. Recently cocrystals of ThiS and ThiG of Bacillus were analysed and intermediates of ThiG catalysis were trapped, giving further insight into the chemistry of thiazole formation (Settembre et al., 2004). These data support the concept of an evolutionary relationship between the pathways of ubiquitin-dependent protein degradation and thiamin as well as molybdenum cofactor biosynthesis, as discussed below.

Molybdenum cofactor

The molybdenum cofactor is required for the activity of a number of molybdoenzymes including nitrate reductase and xanthine dehydrogenase (Hille, 2002). Its biosynthesis is an evolutionarily conserved pathway present in all three kingdoms of life. Only the incorporation of two sulfur atoms into the precursor Z to yield the dithiolene moiety of molybdopterin will be discussed here (Fig. 10). Both sulfur atoms coordinate the molybdenum in the final cofactor structure.

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Figure 10.  Incorporation of two sulfur atoms into precursor Z (left) yields molybdopterin (centre) and enables binding of molybdenum in the final cofactor structure (right).

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At first glance the transformation of precursor Z to molybdopterin seems to have little in common with the formation of thiazole, yet the thiocarboxylate pathway outlined in section Thiamin also applies here. In the following the protein descriptions of E. coli are used as most biochemical data were acquired with this organism. MoaD, an 8.8-kDa protein with the C-terminal Gly–Gly motif is first adenylated by MoeB and details of the interaction of these proteins are known from the X-ray structure of the complex (Lake et al., 2001). Subsequently an IscS-like protein or a rhodanese-like protein converts the MoaD acyl-adenylate to a thiocarboxylate, which acts as the sulfur donor during molybdenum cofactor biosynthesis. Although IscS was able to support an in vitro system of molybdopterin synthesis, iscS mutants of E. coli were able to convert precursor Z to molybdopterin (Leimkühler & Rajagopalan, 2001). The sulfurtransferase involved in vivo therefore remains to be identified. In humans, a cytosolic protein termed MOCS3, which contains a C-terminal rhodanese-like domain, seems to be involved in sulfur transfer to the molybdenum cofactor (Matthies et al., 2004). The MoaE protein incorporates the sulfur of two MoaD thiocarboxylates into precursor Z in two successive steps. Evidence for a reaction intermediate that contains a single sulfur atom has been obtained and a reaction mechanism has been suggested that leaves open whether the attack of the first MoaD thiocarboxylate is at the C-1′ or C-2′ position (Fig. 10) of precursor Z (Wuebbens & Rajagopalan, 2003).

It can be generalized from the sulfur incorporation steps during thiamin as well as molybdopterin biosynthesis that sulfur activation via a thiocarboxylate structure analogous to ubiquitin activation represents a general biochemical reaction module. This module also appears to be used during heterocyst formation of cyanobacteria (Rajagopalan, 1997), in the biosynthesis of sulfur-containing siderophores [pyridine-2,6-bis(thiocarboxylate), quinolobactin] in Pseudomonas species (Lewis et al., 2000; Matthijs et al., 2004), and in a cysteine biosynthetic pathway of Mycobacterium tuberculosis (Burns et al., 2005; Table 2). Further examples will probably have to be added in the future.

Table 2.   Sulfur activation via a thiocarboxylate structure: homologous protein systems
PathwayUbiquitin homologActivating enzyme (E1) homolog
Protein degradationUbiquitinE1/UBA1
Thiamin biosynthesisThiSThiF
Molybdopterin biosynthesisMoaDMoeB
Heterocyst formationHesBHesA
Pyridine-2,6-bis(thiocarboxylate) biosynthesisPdt ORF-HPdt ORF-F
Quinolobactin biosynthesisQbsEQbsC
Cysteine biosynthesisCysOMoeZ

FeS clusters

The biosynthesis of FeS clusters has been reviewed repeatedly and two excellent and very recent reviews cover this topic in great detail (Johnson et al., 2005; Lill & Mühlenhoff, 2005). Therefore, only a conceptional framework is constructed here which elucidates the interaction of the sulfur-activating enzymes introduced above with downstream scaffold or acceptor proteins.

It seems to be a common principle of the nif, isc and suf pathways that activated sulfur and iron are brought together in a cluster precursor hosted by a scaffold protein (Fig. 11). The scaffold protein of the nif pathway is NifU, a modular protein harboring permanent as well as transient FeS clusters. The permanent 2Fe–2S cluster contained in the central NifU domain is suggested to serve a redox role whereas the transient clusters which are assembled in the N- as well as C-terminal domains are both competent to be transferred to aponitrogenase Fe protein in vitro (Smith et al., 2005). Nevertheless, the N-terminal domain has the dominant role in nitrogenase-specific FeS cluster assembly in vivo given that the mutational substitution of cluster ligands in the C-terminal domain did not give rise to a phenotype (Smith et al., 2005). The N-terminal domain of NifU is homologous to the IscU protein of the isc pathway. IscS delivers the activated sulfur to IscU and possibly also to IscA as an alternative scaffold protein. The ferredoxin (Fdx) encoded in the isc-operon seems to be involved in a reduction step with either an iron species or sulfane sulfur as substrate. The Fdx may well be the functional equivalent of the central domain of NifU (Johnson et al., 2005). The chaperones HscA and HscB are a specific requirement of the isc pathway. Although their molecular role is not yet understood in detail, they are involved in some late step of cluster assembly and plausibly have a role in the transfer of cluster precursors from the scaffold proteins to the target FeS proteins (Johnson et al., 2005). The suf machinery is distinct from the nif/isc systems because the assembly route via the scaffold-type protein SufA has not been firmly established. Furthermore, the molecular role of the SufBCD complex comprising the most conserved core components SufB and SufC has not yet been elucidated. SufC is homologous to the ATPase subunit of ABC transporters. SufB and SufD are similar to each other and have been annotated as membrane components of a transporter but the sequences show no indication of intrinsic membrane proteins (Ellis et al., 2001), and the Suf components of E. coli proved to be soluble (Outten et al., 2003). Although the SufBCD complex displays ATPase activity, ATP hydrolysis is not necessary for the enhancement of SufS activity (see section SufS; Outten et al., 2003). Therefore, ATP may be required for some other step of cluster assembly such as iron acquisition. The suf machinery is described in detail in a recent review (Fontecave et al., 2005).

It should be reemphasized that other enzymes generating sulfane sulfur (rhodanese, mercaptopyruvate sulfurtransferase, cystine lyase, l-cysteine desulfidase) have also been implicated in FeS cluster biosynthesis. Because these proteins have not been integrated into the network of a machinery they will not be discussed again here. Finally, it is noteworthy that one IscS-like protein of E. coli belonging to group II, namely CSD, has been described as a minimal cluster assembly machine that works in concert with the SufE equivalent YgdK but in the absence of any scaffold component (Loiseau et al., 2005). Evidently nature has invented more than one method to synthesize the versatile FeS cofactor.

Biotin and lipoic acid

Biotin and lipoic acid are important vitamins that are required as enzyme cofactors in central pathways of cell metabolism. Biotin generally supports carboxylation reactions with pyruvate carboxylase being a standard textbook example. Lipoic acid is involved in acyl transfer reactions and is best known for its role in the pyruvate dehydrogenase complex.

The syntheses of biotin and lipoic acid lead one step beyond FeS cluster bioassembly as FeS clusters play a dual role in the conversion of dethiobiotin to biotin (Jarrett, 2005; Lotierzo et al., 2005) as well as of octanoic acid to lipoic acid (Miller et al., 2000; Ollagnier-de-Choudens et al., 2000; Cicchillo et al., 2004; Cicchillo & Booker, 2005; Fig. 12). Biotin synthase and lipoyl synthase both harbor two FeS clusters per polypeptide. One of the clusters (4Fe–4S) is typical for ‘Adomet radical’ enzymes (see below) and is involved in the activation of the nonactivated C–H bond where the sulfur atom is to be introduced. Sulfide contained in the second cluster (2Fe–2S in biotin synthase, 4Fe–4S in lipoyl synthase) is considered to be the immediate sulfur source (Ugulava et al., 2001; Cicchillo & Booker, 2005). The repair of these clusters through the action of IscS or a related protein would make cysteine the ultimate sulfur source, as suggested for biotin from early studies of labeling (Florentin et al., 1994).

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Figure 12.  Sulfur incorporation reactions of biotin synthase and lipoyl synthase. It should be noted that for the lipoyl synthase of Escherichia coli, the octanoylated derivative of the target protein is the substrate for the sulfur incorporation step (Miller et al., 2000; Zhao et al., 2003; Cicchillo & Booker, 2005).

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The activation of the C–H bond requires adenosylmethionine and a reductant besides the 4Fe–4S cluster, and the mechanism involves radical intermediates. A variety of enzymes using this reaction pathway for quite different purposes have been grouped together as ‘Adomet radical’ or ‘radical SAM’ enzymes; the properties of these enzymes have been reviewed repeatedly, with two very recent reviews dealing with biotin synthase (Jarrett, 2005; Lotierzo et al., 2005). Therefore, no further details are discussed here.

Concerning the sulfur incorporation step it should be noted that the 2Fe–2S cluster is favored as the sulfur source during biotin synthesis at present but alternative possibilities still need to be taken into consideration. It is also possible that the 2Fe–2S cluster serves as a binding site for an exogenous sulfur source, and the involvement of a biotin synthase-based cysteinyl persulfide could even be a possibility (Jarrett, 2005).

Biotin biosynthesis is a fascinating biochemical topic but is also of great biotechnological interest as microbial biotin production would reduce the high environmental burden linked with its chemical synthesis (Streit & Entcheva, 2003). Recombinant strains of B. subtilis as well as E. coli have been described that produce around 1 g of biotin per liter of culture supernatant (Streit & Entcheva, 2003). Although this level of production is nearly profitable, problems with plasmid stability and toxic side-effects still prevent industrial use of this biotechnological process. Perhaps insight into the complex reaction mechanism of biotin synthase will assist the design of better overproducers, which might have an increased need for ‘activated sulfur’.

Thionucleosides (4-thiouridine and 5-methylaminomethyl-2-thiouridine)

Thiolated nucleosides are among the modified nucleosides of tRNA, and E. coli is known to contain four different types of thionucleosides. Their biosyntheses are more or less complicated and not fully elucidated in each case. Nevertheless, studies with E. coli (Lauhon & Kambampati, 2000; Lauhon, 2002) and S. enterica serovar Typhimurium (Leipuviene et al., 2004) have established that in vivo sulfur activated by IscS is incorporated into 4-thiouridine via ThiI and into 2-thiouridine via MnmA (formerly also named AsuE or TrmU); additional enzymatic steps convert 2-thiouridine to the final product 5-methylaminomethyl-2-thiouridine (Fig. 13). The biosyntheses of the remaining thionucleosides require enzymes considered to be FeS proteins, complicating the interpretation of the role of IscS in these cases (see Pierrel et al., 2004).

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Figure 13.  Biosynthesis of the tRNA thionucleosides 4-thiouridine and 5-methylaminomethyl-2-thiouridine in Escherichia coli.

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Although mechanistic details remain to be determined it is clear that the sulfur chemistry outlined for thiamin biosynthesis also applies here, at least for ThiI-assisted 4-thiouridine synthesis in E. coli. Sulfur activated by IscS is transferred to the rhodanese-like module of ThiI (Palenchar et al., 2000). An analogous step for MnmA that does not contain a rhodanese-like module is less clear because a persulfidic intermediate could not be observed with this protein but has been suggested (Kambampati & Lauhon, 2003). Additional proteins involved in the MnmA pathway have recently been identified that constitute a complex sulfur-relay system. They are termed TusA, TusBCD and TusE; TusA, TusD and TusE were characterized as persulfide carriers (Ikeuchi et al., 2006). An enzyme-persulfide is therefore the most probable sulfur donor to the activated uridine for 4-thiouridine as well as 2-thiouridine formation. Most probably, activation of the uridine is carried out by adenylation of the oxygen atom to be substituted by sulfur. The adenylation reaction may also be catalysed by ThiI (Palenchar et al., 2000) or MnmA (Kambampati & Lauhon, 2003), respectively. They share similar nucleotide binding loop sequences (Kambampati & Lauhon, 2003) and require ATP for activity.

I have already mentioned in section IscS that IscS is the central sulfur-providing enzyme in E. coli, and the details presented here and in the other biosynthetic subsections corroborate this statement. The interesting question then arises of whether certain properties of IscS can be identified that are of specific importance for only one pathway or a subset of pathways. This question has been addressed by alanine-scanning mutagenesis of the IscS active-site loop residues (Lauhon et al., 2004). Two mutant strains were identified that were severely impaired in FeS cluster biosynthesis but showed wild-type levels of 4-thiouridine and 5-methylaminomethyl-2-thiouridine (Lauhon et al., 2004). Therefore, FeS cluster biosynthesis might be the most elaborate pathway involving IscS in E. coli. In vitro the two IscS mutant proteins were similar to wild-type IscS with regard to their desulfurase activity and sulfur transfer to IscU (Lauhon et al., 2004). Evidently, additional in vitro assays will have to be developped to examine the role of IscS in FeS cluster assembly down to the last detail.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References

Cysteine is certainly the central metabolite of sulfur biochemistry. Its biosynthesis requires the incorporation of sulfide, which is therefore available inside cells. Nevertheless, a variety of cysteine desulfurating enzymes exist that separate the incorporated sulfur from the cysteine carbon skeleton to produce persulfidic sulfur. This sulfur species – which can also be produced from substrates other than cysteine – supplies a number of important and elaborate pathways requiring ‘activated sulfur’. Why did nature chose to use persulfides instead of sulfide in so many pathways, among them fundamental and therefore putatively ancient ones?. Toxicity is often an argument used to exclude sulfide. But persulfides are also highly reactive and toxic effects have been described (Gentry-Weeks et al., 1993; Krupka et al., 2000; see section Cystinelyase). In addition, sulfide-dependent pathways are tolerated by cells. The advantage may lie in the specific chemical properties of persulfidic sulfur: being bonded to some larger molecule it can be handled more specifically than sulfide. More molecular interactions are possible, especially when the persulfide moiety is carried by a protein. Given the ease of transfer of persulfidic sulfur to thiol acceptors, sulfur relay systems are functional and deliver the active sulfur to specific target sites. Redox reactions of persulfides with thiol–disulfide redox couples are feasible and allow liberation of sulfide when finally required. Persulfides may therefore be regarded as ‘sulfide with a handle’ as with ATP, which presents ‘phosphate with a handle’, a comparison that has already been made by Beinert (2000a). More and more pathways are being discovered that involve ‘active sulfur’ in the form of persulfides instead of sulfide in vivo, and even those reactions that until now have been considered to use sulfide may turn out to require persulfidic sulfur as sulfide precursor. It will be interesting to see whether additional enzymological and genetic studies will support the concept of persulfides as the general sulfur currency of the cell.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sulfur-containing biomolecules
  5. Chemical properties of persulfidic sulfur
  6. Enzymes generating persulfidic sulfur
  7. Specific biosynthetic pathways using ‘activated sulfur’
  8. Concluding remarks
  9. Acknowledgements
  10. References
  • Adams H, Teertstra W, Koster M & Tommassen J (2002) PspE (phage-shock protein E) of Escherichia coli is a rhodanese. FEBS Lett 518: 173176.
  • Ali V, Shigeta Y, Tokumoto U, Takahashi Y & Nozaki T (2004) An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions. J Biol Chem 279: 1686316874.
  • Bartsch RG, Newton GL, Sherrill C & Fahey RC (1996) Glutathione amide and its perthiol in anaerobic sulfur bacteria. J Bacteriol 178: 47424746.
  • Begley TP, Downs DM, Ealick SE et al. (1999) Thiamin biosynthesis in prokaryotes. Arch Microbiol 171: 293300.
  • Behshad E, Parkin SE & Bollinger JM (2004) Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: kinetic analysis for cleavage of the persulfide intermediate by chemical reductants. Biochemistry 43: 1222012226.
  • Beinert H (2000a) A tribute to sulfur. Eur J Biochem 267: 56575664.
  • Beinert H (2000b) Iron–sulfur proteins: ancient structures, still full of surprises. J Biol Inorg Chem 5: 215.
  • Bordo D & Bork P (2002) The rhodanese/Cdc25 phosphatase superfamily. Sequence-structure-function relations. EMBO Reports 3: 741746.
  • Burns KE, Baumgart S, Dorrestein PC, Zhai H, McLafferty FW & Begley TP (2005) Reconstitution of a new cysteine biosynthetic pathway in Mycobacterium tuberculosis. J Am Chem Soc 127: 1160211603.
  • Cavallini D, De Marco C, Mondovi B & Mori BG (1960) The cleavage of cystine by cystathionase and the transulfuration of hypotaurine. Enzymologia 22: 161173.
  • Cavallini D, Federici G & Barboni E (1970) Interaction of proteins with sulfide. Eur J Biochem 14: 169174.
  • Cerletti P (1986) Seeking a better job for an under-employed enzyme: rhodanese. Trends Biochem Sci 11: 369372.
  • Cianci M, Gliubich F, Zanotti G & Berni R (2000) Specific interaction of lipoate at the active site of rhodanese. Biochim Biophys Acta 1481: 103108.
  • Cicchillo RM & Booker SJ (2005) Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide. J Am Chem Soc 127: 28602861.
  • Cicchillo RM, Iwig DF, Jones AD, Nesbitt NM, Baleanu-Gogonea C, Souder MG, Tu L & Booker SJ (2004) Lipoyl synthase requires two equivalents of S-adenosyl-l-methionine to synthesize one equivalent of lipoic acid. Biochemistry 43: 63786386.
  • Clausen T, Kaiser JT, Steegborn C, Huber R & Kessler D (2000) Crystal structure of the cystine C-S lyase from Synechocystis: Stabilization of cysteine persulfide for FeS cluster biosynthesis. Proc Natl Acad Sci USA 97: 38563861.
  • Cupp-Vickery JR, Urbina H & Vickery LE (2003) Crystal structure of IscS, a cysteine desulfurase from Escherichia coli. J Mol Biol 330: 10491059.
  • Delavier-Klutchko C & Flavin M (1965) Role of a bacterial cystathionine β-cleavage enzyme in disulfide decomposition. Biochim Biophys Acta 99: 375377.
  • Ding H, Harrison K & Lu J (2005) Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU. J Biol Chem 280: 3043230437.
  • Donadio S, Shafiee A & Hutchinson CR (1990) Disruption of a rhodaneselike gene results in cysteine auxotrophy in Saccharopolyspora erythraea. J Bacteriol 172: 350360.
  • Ellis KES, Clough B, Saldanha JW & Wilson RJM (2001) Nifs and Sufs in malaria. Mol Microbiol 41: 973981.
  • Everett SA & Wardman P (1995) Perthiols as antioxidants: radical-scavenging and prooxidative mechanisms. Meth Enzymol 251: 5569.
  • Flavin M (1962) Microbial transsulfuration: the mechanism of an enzymatic disulfide elimination reaction. J Biol Chem 237: 768777.
  • Flint DH, Tuminello JF & Miller TJ (1996) Studies on the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase in Escherichia coli crude extract. Isolation of O-acetylserine sulfhydrylases A and B and β-cystathionase based on their ability to mobilize sulfur from cysteine and to participate in Fe–S cluster synthesis. J Biol Chem 271: 1605316067.
  • Florentin D, Tse Sum Bui B, Marquet A, Ohshiro T & Izumi Y (1994) On the mechanism of biotin synthase of Bacillus sphaericus. CR Acad Sci Paris Life Sci 317: 485488.
  • Fontecave M, Ollagnier-de-Choudens S, Py B & Barras F (2005) Mechanisms of iron-sulfur cluster assembly: the SUF machinery. J Biol Inorg Chem 10: 713721.
  • Fujii T, Maeda M, Mihara H, Kurihara T, Esaki N & Hata Y (2000) Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase. Biochemistry 39: 12631273.
  • Gentry-Weeks CR, Keith JM & Thompson J (1993) Toxicity of Bordetella aviumβ-cystathionase toward MC3T3-E1 osteogenic cells. J Biol Chem 268: 72987314.
  • Goldsmith-Fischman S, Kuzin A, Edstrom WC, Benach J, Shastry R, Xiao R, Acton TB, Honig B, Montelione GT & Hunt JF (2004) The SufE sulfur-acceptor protein contains a conserved core structure that mediates interdomain interactions in a variety of redox protein complexes. J Mol Biol 344: 549565.
  • Graham DE & White RH (2002) Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics. Nat Prod Rep 19: 133147.
  • Hand CE & Honek JF (2005) Biological chemistry of naturally occurring thiols of microbial and marine origin. J Nat Prod 68: 293308.
  • Hille R (2002) Molybdenum and tungsten in biology. Trends Biochem Sci 27: 360367.
  • Huet G, Daffé M & Saves I (2005) Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe–S] cluster assembly: evidence for its implication in the pathogen's survival. J Bacteriol 187: 61376146.
  • Ikeuchi Y, Shigi N, Kato J, Nishimura A & Suzuki T (2006) Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol Cell 21: 97108.
  • Jarabak R & Westley J (1980) 3-Mercaptopyruvate sulfurtransferase: rapid equilibrium-ordered mechanism with cyanide as the acceptor substrate. Biochemistry 19: 900904.
  • Jarrett JT (2005) The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase. Arch Biochem Biophys 433: 312321.
  • Johnson D, Dean DR, Smith AD & Johnson MK (2005) Structure, function, and formation of biological iron–sulfur clusters. Annu Rev Biochem 74: 247281.
  • Kaiser JT, Clausen T, Bourenkow GP, Bartunik H-D, Steinbacher S & Huber R (2000) Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly. J Mol Biol 297: 451464.
  • Kaiser JT, Bruno S, Clausen T, Huber R, Schiaretti F, Mozzarelli A & Kessler D (2003) Snapshots of the cystine lyase C-DES during catalysis. Studies in solution and in the crystalline state. J Biol Chem 278: 357365.
  • Kambampati R & Lauhon CT (2003) MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry 42: 11091117.
  • Kessler D (2004) Slr0077 of Synechocystis has cysteine desulfurase as well as cystine lyase activity. Biochem Biophys Res Commun 320: 571577.
  • Kleerebezem M, Crielaard W & Tommassen J (1996) Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions. EMBO J 15: 162171.
  • Krupka HI, Huber R, Holt SC & Clausen T (2000) Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5′-phosphate-dependent protein acting as a haemolytic enzyme. EMBO J 19: 31683178.
  • Lake MW, Wuebbens MM, Rajagopalan KV & Schindelin H (2001) Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414: 325329.
  • Lang T & Kessler D (1999) Evidence for cysteine persulfide as reaction product of l-cyst(e)ine C-S-lyase (C-DES) from Synechocystis. Analyses using cystine analogues and recombinant C-DES. J Biol Chem 274: 189195.
  • Lauhon CT (2002) Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli. J Bacteriol 184: 68206829.
  • Lauhon C & Kambampati R (2000) The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD. J Biol Chem 275: 2009620103.
  • Lauhon CT, Skovran E, Urbina HD, Downs DM & Vickery LE (2004) Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo. J Biol Chem 279: 1955119558.
  • Lee J-H, Yeo W-S & Roe J-H (2004) Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor. Mol Microbiol 51: 17451755.
  • Leibrecht I & Kessler D (1997) A novel l-cysteine/cystine C-S lyase directing[2Fe–2S] cluster formation of Synechocystis ferredoxin. J Biol Chem 272: 1044210447.
  • Leimkühler S & Rajagopalan KV (2001) A sulfurtransferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z in Escherichia coli. J Biol Chem 276: 2202422031.
  • Leipuviene R, Qian Q & Bjork GR (2004) Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. J Bacteriol 186: 758766.
  • Lewis TA, Cortese MS, Sebat JL, Green TL, Lee C-H & Crawford RL (2000) A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid). Environ Microbiol 2: 407416.
  • Lill R & Mühlenhoff U (2005) Iron–sulfur-protein biogenesis in eukaryotes. Trends Biochem Sci 30: 133141.
  • Lima CD (2002) Analysis of the E. coli NifS CsdB protein at 2.0 Å reveals the structural basis for perselenide and persulfide intermediate formation. J Mol Biol 315: 11991208.
  • Loiseau L, Ollagnier-de-Choudens S, Nachin L, Fontecave M & Barras F (2003) Biogenesis of Fe–S cluster by the bacterial Suf system. SufS and SufE form a new type of cysteine desulfurase. J Biol Chem 278: 3835238359.
  • Loiseau L, Ollagnier-de-Choudens S, Lascoux D, Forest E, Fontecave M & Barras F (2005) Analysis of the heteromeric CsdA–CsdE cysteine desulfurase, assisting Fe–S cluster biogenesis in Escherichia coli. J Biol Chem 280: 2676026769.
  • Lotierzo M, Tse Sum Bui B, Florentin D, Escalettes F & Marquet A (2005) Biotin synthase mechanism: an overview. Biochem Soc Trans 33: 820823.
  • Matthies A, Rajagopalan KV, Mendel RR & Leimkühler S (2004) Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans. Proc Natl Acad Sci USA 101: 59465951.
  • Matthijs S, Baysse C, Koedam N et al. (2004) The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway. Mol Microbiol 52: 371384.
  • Mihara H, Kurihara T, Yoshimura T, Soda K & Esaki N (1997) Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme. J Biol Chem 272: 2241722424.
  • Mihara H, Maeda M, Fujii T, Kurihara T, Hata Y & Esaki N (1999) A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary X-ray crystallographic studies. J Biol Chem 274: 1476814772.
  • Miller JR, Busby RW, Jordan SW, Cheek J, Henshaw TF, Ashley GW, Broderick JB, Cronan JE Jr & Marletta MA (2000) Escherichia coli LipA is a lipoyl synthase: "in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. Biochemistry 39: 1516615178.
  • Newton GL & Javor B (1985) γ-Glutamylcysteine and thiosulfate are the major low-molecular weight thiols in Halobacteria. J Bacteriol 161: 438441.
  • Ollagnier-de-Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, Münck E & Fontecave M (2000) Iron–sulfur center of biotin synthase and lipoate synthase. Biochemistry 39: 41654173.
  • Ollagnier-de-Choudens S, Lascoux D, Loiseau L, Barras F, Forest E & Fontecave M (2003) Mechanistic studies of the SufS–SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE. FEBS Lett 555: 263267.
  • Olson JW, Agar JN, Johnson MK & Maier RJ (2000) Characterization of the NifU and NifS Fe–S cluster formation proteins essential for viability in Helicobacter pylori. Biochemistry 39: 1621316219.
  • Outten FW, Wood MJ, Muñoz M & Storz G (2003) The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe–S cluster assembly in Escherichia coli. J Biol Chem 278: 4571345719.
  • Outten FW, Djaman O & Storz G (2004) A suf operon requirement for Fe–S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol 52: 861872.
  • Palenchar PM, Buck CJ, Cheng H, Larson TJ & Mueller EG (2000) Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate. J Biol Chem 275: 82838286.
  • Park J-H, Dorrestein PC, Zhai H, Kinsland C, McLafferty FW & Begley TP (2003) Biosynthesis of the thiazole moiety of thiamin pyrophosphate (vitamin B1). Biochemistry 42: 1243012438.
  • Park S & Imlay JA (2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J Bacteriol 185: 19421950.
  • Patzer SI & Hantke K (1999) SufS is a NifS-like protein, and SufD is necessary for stability of the[2Fe–2S] FhuF protein in Escherichia coli. J Bacteriol 181: 33073309.
  • Pierrel F, Douki T, Fontecave M & Atta M (2004) MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA. J Biol Chem 279: 4755547563.
  • Rajagopalan KV (1997) Biosynthesis and processing of the molybdenum cofactors. Biochem Soc Trans 25: 757761.
  • Ravot G, Casalot L, Ollivier B, Loison G & Magot M (2005) rdlA, a new gene encoding a rhodanese-like protein in Halanerobium congolense and other thiosulfate-reducing anaerobes. Res Microbiol 156: 10311038.
  • Ray WK, Zeng G, Potters MB, Mansuri AM & Larson TJ (2000) Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin. J Bacteriol 182: 22772284.
  • Rohwerder T & Sand W (2003) The sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes from Acidithiobacillus and Acidiphilium spp.. Microbiology 149: 16991709.
  • Sauerwald A, Zhu W, Major TA, Roy H, Palioura S, Jahn D, Whitman WB, Yates JR III, Ibba M & Söll D (2005) RNA-dependent cysteine biosynthesis in Archaea. Science 307: 19691972.
  • Schmidt A & Jäger K (1992) Open questions about sulfur metabolism in plants. Annu Rev Plant Physiol Plant Mol Biol 43: 325349.
  • Settembre EC, Dorrestein PC, Zhai H, Chatterjee A, McLafferty FW, Begley TP & Ealick SE (2004) Thiamin biosynthesis in Bacillus subtilis: structure of the thiazole synthase/sulfur carrier protein complex. Biochemistry 43: 1164711657.
  • Skovran E & Downs DM (2000) Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar Typhimurium: implications for thiamine synthesis. J Bacteriol 182: 38963903.
  • Smith AD, Jameson GNL, DosSantos PC, Agar JN, Naik S, Krebs C, Frazzon J, Dean DR, Huynh BH & Johnson MK (2005) NifS-mediated assembly of[4Fe–4S] clusters in the N- and C-terminal domains of the NifU scaffold protein. Biochemistry 44: 1295512969.
  • Spallarossa A, Forlani F, Carpen A, Armirotti A, Pagani S, Bolognesi M & Bordo D (2004) The ‘rhodanese’ fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferase: crystal structure of SseA from Escherichia coli. J Mol Biol 335: 583593.
  • Steegborn C, Clausen T, Sondermann P, Jacob U, Worbs M, Marinkovic S, Huber R & Wahl MC (1999) Kinetics and inhibition of recombinant human cystathionine γ-lyase. Toward the rational control of transsulfuration. J Biol Chem 274: 1267512684.
  • Streit WR & Entcheva P (2003) Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production. Appl Microbiol Biotechnol 61: 2131.
  • Tachezy J, Sánchez LB & Müller M (2001) Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. Mol Biol Evol 18: 19191928.
  • Takahashi Y & Tokumoto U (2002) A third bacterial system for the assembly of iron–sulfur clusters with homologs in Archaea and plastids. J Biol Chem 277: 2838028383.
  • Taylor SV, Kelleher NL, Kinsland C, Chiu H-J, Costello CA, Backstrom AD, McLafferty FW & Begley TP (1998) Thiamin biosynthesis in Escherichia coli. Identification of ThiS thiocarboxylate as the immediate sulfur donor in the thiazole formation. J Biol Chem 273: 1655516560.
  • Tchong S, Xu H & White RH (2005) L-Cysteine desulfidase: an [4Fe–4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia and sulfide. Biochemistry 44: 16591670.
  • Theissen U, Hoffmeister M, Grieshaber M & Martin W (2003) Single eubacterial origin of eukaryotic sulfide: uinone oxidoreductase, a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times. Mol Biol Evol 20: 15641574.
  • Tirupati B, Vey JL, Drennan CL & Bollinger JM (2004) Kinetic and structural characterization of Slr0077/SufS, the essential cysteine desulfurase from Synechocystis sp. PCC 6803. Biochemistry 43: 1221012219.
  • Toohey JI (1989) Sulphane sulphur in biological systems: a possible regulatory role. Biochem J 264: 625632.
  • Tovar J, León-Avila G, Sánchez LB, Sutak R, Tachezy J, Van Der Giezen M, Hernández M, Müller M & Lucocq JM (2003) Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 426: 172176.
  • Ugulava NB, Sacanell CJ & Jarrett JT (2001) Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe–2S] cluster accompanies sulfur insertion. Biochemistry 40: 83528358.
  • Vamvakas S, Berthold K, Dekant W & Henschler D (1988) Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine-S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates. Chem Biol Interact 65: 5971.
  • Wang R (2002) Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 16: 17921798.
  • Webb E, Claas K & Downs DM (1997) Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium. J Bacteriol 179: 43994402.
  • Wolfe MD, Ahmed F, Lacourciere GM, Lauhon CT, Stadtman TC & Larson TJ (2004) Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase. J Biol Chem 279: 18011809.
  • Wood JL (1987) Sulfane sulfur. Meth Enzymol 143: 2529.
  • Wuebbens MM & Rajagopalan KV (2003) Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis. J Biol Chem 278: 1452314532.
  • Xi J, Ge Y, Kinsland C, McLafferty FW & Begley TP (2001) Biosynthesis of the thiazole moiety of thiamin in Escherichia coli: identification of an acyldisulfide-linked protein–protein conjugate that is functionally analogous to the ubiquitin/E1 complex. Proc Natl Acad Sci USA 98: 85138518.
  • Zdych E, Peist R, Reidl J & Boos W (1995) MalY of Escherichia coli is an enzyme with the activity of a βC-S lyase (cystathionase). J Bacteriol 177: 50355039.
  • Zhao S, Miller JR, Jiang Y, Marletta MA & Cronan JE Jr (2003) Assembly of the covalent linkage between lipoic acid and its cognate enzymes. Chem Biol 10: 12931302.
  • Zheng L, White RH, Cash VL, Jack RF & Dean DR (1993) Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci USA 90: 27542758.
  • Zheng L, White RH, Cash VL & Dean DR (1994) Mechanism for the desulfurization of L-cysteine catalyzed by the nifS gene product. Biochemistry 33: 47144720.
  • Zheng L, Cash VL, Flint DH & Dean DR (1998) Assembly of iron–sulfur clusters. Identification of an iscSUA–hscBA–fdx gene cluster from Azotobacter vinelandii. J Biol Chem 273: 1326413272.
  • Zheng M, Wang X, Templeton LJ, Smulski DR, LaRossa RA & Storz G (2001) DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 183: 45624570.