Download Enzymatic activation of sulfur for incorporation into biomolecules in

Enzymatic activation of sulfur for incorporation into biomolecules
in prokaryotes
Dorothea Kessler
Biochemiezentrum Heidelberg, Universität Heidelberg, Heidelberg, Germany
Correspondence: Dorothea Kessler,
Biochemiezentrum Heidelberg, Universität
Heidelberg, Im Neuenheimer Feld 328,
D-69120 Heidelberg, Germany. Tel.: 1 49
6221 544289; fax:149 6221 544769; e-mail:
[email protected]
Received 28 February 2006; revised 19 May
2006; accepted: 22 May 2006.
First published online 17 August 2006.
DOI:10.1111/j.1574-6976.2006.00036.x
Editor: Fritz Unden
Abstract
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.
Keywords
persulfide; sulfurtransferase; desulfurase;
cystine lyase; prokaryotic sulfur metabolism;
ubiquitin.
Introduction
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.
FEMS Microbiol Rev 30 (2006) 825–840
Sulfur-containing biomolecules
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 mM–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 mM (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 mM (Park & Imlay, 2003).
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
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826
D. Kessler
Fig. 2. Sulfur compounds relying on persulfidic sulfur (R-S-SH) as sulfur
source.
Fig. 1. Biosynthesis of cysteine and overview of sulfur-containing biomolecules based on cysteine. THF, tetrahydrofolate.
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 g-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 tRNAdependent. In a first step tRNACys becomes acylated with
O-phosphoserine; in a second step O-phosphoserinetRNACys 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 biologi2006 Federation of European Microbiological Societies
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cally 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.
Chemical properties of persulfidic sulfur
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).
R-S-SH ! R-SH þ S:
ð1Þ
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 kJM1 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 M1 cm1, Cavallini et al.,
1970; 310 M1 cm1, 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.
R-S-SH þ CN ! R-SH þ SCN :
ð2Þ
FEMS Microbiol Rev 30 (2006) 825–840
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Enzymatic activation of sulfur in prokaryotes
In addition to persulfides, other sulfane sulfur compounds such as thiosulfate (SO3S), thiosulfonate ion
(RSO3S), polysulfides (RSSnSR) or polythionates
(SO3SnSO
3 ) 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):
þ
GSSH þ O2 þ H2 O ! GSH þ SO2
3 þ 2H :
ð3Þ
The glutathione persulfide was formed in situ in the
reaction mixtures either from glutathione and elemental
sulfur (GSH1S0 ! GSSH) or from glutathione disulfide
and hydrogen sulfide (GSSG1H2S ! GSSH1GSH). 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-molecularweight thiol of Halobacteria again represents a sulfane sulfur
compound although a more stable one: thiosulfate was
found in these cells besides g-glutamylcysteine which is
synthesized instead of glutathione in Halobacteria. It was
FEMS Microbiol Rev 30 (2006) 825–840
shown that thiosulfate stems from oxidation of cysteine
(Newton & Javor, 1985).
In summary it can be concluded that free low-molecularweight 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
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-XGly 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
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828
D. Kessler
IscS
Fig. 3. Cleavage of the C – S bond of cysteine bound to the pyridoxal
phosphate cofactor of NifS.
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 mg1 (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.
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 afamily 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.
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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 0 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
FEMS Microbiol Rev 30 (2006) 825–840
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Enzymatic activation of sulfur in prokaryotes
Fig. 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.
Table 1. Designations and classification of cysteine desulfurases
Name
Group
Synonyms
NifS
IscS
SufS
I
I
II
CSD (cysteine sulfinate
desulfinase)
II
–
Slr0387
CsdB (selenocysteine
lyase); Slr0077
CsdA
Slr, Synechocystis open reading frame longer than 100 codons, reading
direction right.
(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.
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 mg1 (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 s1
was determined using Synechocystis SufS (Tirupati et al.,
FEMS Microbiol Rev 30 (2006) 825–840
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).
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
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830
D. Kessler
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 enzymebased 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):
2
SSO2
3 þ Rhodanese-SH ! SO3
þ Rhodanese-S-SH;
ð4aÞ
Rhodanese-S-SH þ CN ! Rhodanese-SH þ SCN :
ð4bÞ
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
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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 singledomain rhodanese of E. coli, PspE, is a periplasmic protein
(Adams et al., 2002). Its gene belongs to the psp (phageshock 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 2thiouridine 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:
ðHSÞH2 C-CO-CO
2 þ CN ! H3 C-CO-CO2 þ SCN :
ð5Þ
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
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Enzymatic activation of sulfur in prokaryotes
Fig. 5. Disulfide – thiosulfoxide isomerization reaction suggested to
occur in the active site of the Escherichia coli mercaptopyruvate sulfurtransferase SseA.
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).
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.
This activity has been long established as a side-activity of
cystathionase from rat liver (Cavallini et al., 1960), gcystathionase from Neurospora (Flavin, 1962) or b-cystathionase from E. coli (Delavier-Klutchko & Flavin,
1965). The b-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 g-cystathionase, b-cystathionase
and cystathionine g-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 apoto 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 CDES, 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
b-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
Cystine lyase
The cystine lyase reaction represents a pyridoxal phosphatebased b-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):
OOC CHðNHþ
3 Þ CH2 S SH
cysteine persulfide
ð6Þ
þ pyruvate þ NHþ
4:
Cystine þ H2 O !
FEMS Microbiol Rev 30 (2006) 825–840
Fig. 6. Active-site residues of the cystine lyase C-DES and interaction
with the products of cystine cleavage (dashed lines).
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832
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.
Cystine lyase activity is also associated with some other
proteins of microbiological interest, even though its significance is not completely understood: cysteine conjugate
b-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 b-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 belimination, 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 b-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
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D. Kessler
Fig. 7. Proposed generation of cluster-bound sulfide by L-cysteine desulfidase.
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.
Specific biosynthetic pathways using
‘activated sulfur’
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).
The crucial sulfur-containing intermediate is the ThiS
protein thiocarboxylate, which was elucidated from the
E. coli system (Taylor et al., 1998). Alternatively, the
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Enzymatic activation of sulfur in prokaryotes
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 Cterminal 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.
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-5phosphate 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
Fig. 8. Generation of ThiS thiocarboxylate as sulfur donor for thiazole
formation in Escherichia coli.
Fig. 9. Mechanistic suggestion for IscS and ThiI action during ThiS – ThiF
acyldisulfide formation in Escherichia coli.
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.
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 8.8kDa 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
Fig. 10. Incorporation of two sulfur atoms into precursor Z (left) yields molybdopterin (centre) and enables binding of molybdenum in the final cofactor
structure (right).
FEMS Microbiol Rev 30 (2006) 825–840
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D. Kessler
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 0 or C-2 0 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.
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 sulfuractivating enzymes introduced above with downstream
scaffold or acceptor proteins.
Table 2. Sulfur activation via a thiocarboxylate structure: homologous
protein systems
Pathway
Protein degradation
Thiamin biosynthesis
Molybdopterin biosynthesis
Heterocyst formation
Pyridine-2,6-bis(thiocarboxylate)
biosynthesis
Quinolobactin biosynthesis
Cysteine biosynthesis
Ubiquitin
homolog
Activating enzyme (E1)
homolog
Ubiquitin
ThiS
MoaD
HesB
Pdt ORF-H
E1/UBA1
ThiF
MoeB
HesA
Pdt ORF-F
QbsE
CysO
QbsC
MoeZ
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Fig. 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).
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
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Enzymatic activation of sulfur in prokaryotes
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).
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)
Fig. 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).
FEMS Microbiol Rev 30 (2006) 825–840
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
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D. Kessler
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.
Fig. 13. Biosynthesis of the tRNA thionucleosides 4-thiouridine and 5methylaminomethyl-2-thiouridine in Escherichia coli.
Concluding remarks
2-thiouridine to the final product 5-methylaminomethyl-2thiouridine (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).
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 4thiouridine 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
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.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Microbiol Rev 30 (2006) 825–840
837
Enzymatic activation of sulfur in prokaryotes
Acknowledgements
I wish to thank Dr Jennifer Ortiz (Biochemiezentrum
Heidelberg) for thoughtfully improving the English of the
manuscript.
References
Adams H, Teertstra W, Koster M & Tommassen J (2002) PspE
(phage-shock protein E) of Escherichia coli is a rhodanese.
FEBS Lett 518: 173–176.
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:
16863–16874.
Bartsch RG, Newton GL, Sherrill C & Fahey RC (1996)
Glutathione amide and its perthiol in anaerobic sulfur
bacteria. J Bacteriol 178: 4742–4746.
Begley TP, Downs DM, Ealick SE et al. (1999) Thiamin
biosynthesis in prokaryotes. Arch Microbiol 171: 293–300.
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: 12220–12226.
Beinert H (2000a) A tribute to sulfur. Eur J Biochem 267:
5657–5664.
Beinert H (2000b) Iron–sulfur proteins: ancient structures, still
full of surprises. J Biol Inorg Chem 5: 2–15.
Bordo D & Bork P (2002) The rhodanese/Cdc25 phosphatase
superfamily. Sequence-structure-function relations. EMBO
Reports 3: 741–746.
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:
11602–11603.
Cavallini D, De Marco C, Mondovi B & Mori BG (1960) The
cleavage of cystine by cystathionase and the transulfuration of
hypotaurine. Enzymologia 22: 161–173.
Cavallini D, Federici G & Barboni E (1970) Interaction of
proteins with sulfide. Eur J Biochem 14: 169–174.
Cerletti P (1986) Seeking a better job for an under-employed
enzyme: rhodanese. Trends Biochem Sci 11: 369–372.
Cianci M, Gliubich F, Zanotti G & Berni R (2000) Specific
interaction of lipoate at the active site of rhodanese. Biochim
Biophys Acta 1481: 103–108.
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: 2860–2861.
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:
6378–6386.
FEMS Microbiol Rev 30 (2006) 825–840
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: 3856–3861.
Cupp-Vickery JR, Urbina H & Vickery LE (2003) Crystal
structure of IscS, a cysteine desulfurase from Escherichia coli.
J Mol Biol 330: 1049–1059.
Delavier-Klutchko C & Flavin M (1965) Role of a bacterial
cystathionine b-cleavage enzyme in disulfide decomposition.
Biochim Biophys Acta 99: 375–377.
Ding H, Harrison K & Lu J (2005) Thioredoxin reductase system
mediates iron binding in IscA and iron delivery for the ironsulfur cluster assembly in IscU. J Biol Chem 280: 30432–30437.
Donadio S, Shafiee A & Hutchinson CR (1990) Disruption of a
rhodaneselike gene results in cysteine auxotrophy in
Saccharopolyspora erythraea. J Bacteriol 172: 350–360.
Ellis KES, Clough B, Saldanha JW & Wilson RJM (2001) Nifs and
Sufs in malaria. Mol Microbiol 41: 973–981.
Everett SA & Wardman P (1995) Perthiols as antioxidants:
radical-scavenging and prooxidative mechanisms. Meth
Enzymol 251: 55–69.
Flavin M (1962) Microbial transsulfuration: the mechanism of an
enzymatic disulfide elimination reaction. J Biol Chem 237:
768–777.
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 b-cystathionase based on their
ability to mobilize sulfur from cysteine and to participate in
Fe–S cluster synthesis. J Biol Chem 271: 16053–16067.
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: 485–488.
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: 713–721.
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: 1263–1273.
Gentry-Weeks CR, Keith JM & Thompson J (1993) Toxicity of
Bordetella avium b-cystathionase toward MC3T3-E1
osteogenic cells. J Biol Chem 268: 7298–7314.
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: 549–565.
Graham DE & White RH (2002) Elucidation of methanogenic
coenzyme biosyntheses: from spectroscopy to genomics. Nat
Prod Rep 19: 133–147.
Hand CE & Honek JF (2005) Biological chemistry of naturally
occurring thiols of microbial and marine origin. J Nat Prod 68:
293–308.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
838
Hille R (2002) Molybdenum and tungsten in biology. Trends
Biochem Sci 27: 360–367.
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:
6137–6146.
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: 97–108.
Jarabak R & Westley J (1980) 3-Mercaptopyruvate
sulfurtransferase: rapid equilibrium-ordered mechanism with
cyanide as the acceptor substrate. Biochemistry 19: 900–904.
Jarrett JT (2005) The novel structure and chemistry of iron-sulfur
clusters in the adenosylmethionine-dependent radical enzyme
biotin synthase. Arch Biochem Biophys 433: 312–321.
Johnson D, Dean DR, Smith AD & Johnson MK (2005) Structure,
function, and formation of biological iron–sulfur clusters.
Annu Rev Biochem 74: 247–281.
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: 451–464.
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: 357–365.
Kambampati R & Lauhon CT (2003) MnmA and IscS are
required for in vitro 2-thiouridine biosynthesis in Escherichia
coli. Biochemistry 42: 1109–1117.
Kessler D (2004) Slr0077 of Synechocystis has cysteine desulfurase
as well as cystine lyase activity. Biochem Biophys Res Commun
320: 571–577.
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: 162–171.
Krupka HI, Huber R, Holt SC & Clausen T (2000) Crystal
structure of cystalysin from Treponema denticola: a pyridoxal
5 0 -phosphate-dependent protein acting as a haemolytic
enzyme. EMBO J 19: 3168–3178.
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:
325–329.
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: 189–195.
Lauhon CT (2002) Requirement for IscS in biosynthesis of all
thionucleosides in Escherichia coli. J Bacteriol 184: 6820–6829.
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: 20096–20103.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
D. Kessler
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: 19551–19558.
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:
1745–1755.
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: 10442–10447.
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: 22024–22031.
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: 758–766.
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-2,6bis(thiocarboxylic acid). Environ Microbiol 2: 407–416.
Lill R & Mühlenhoff U (2005) Iron–sulfur-protein biogenesis in
eukaryotes. Trends Biochem Sci 30: 133–141.
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: 1199–1208.
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: 38352–38359.
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: 26760–26769.
Lotierzo M, Tse Sum Bui B, Florentin D, Escalettes F & Marquet A
(2005) Biotin synthase mechanism: an overview. Biochem Soc
Trans 33: 820–823.
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: 5946–5951.
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: 371–384.
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:
22417–22424.
FEMS Microbiol Rev 30 (2006) 825–840
839
Enzymatic activation of sulfur in prokaryotes
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: 14768–14772.
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 octanoylacyl carrier protein. Biochemistry 39: 15166–15178.
Newton GL & Javor B (1985) g-Glutamylcysteine and thiosulfate
are the major low-molecular weight thiols in Halobacteria.
J Bacteriol 161: 438–441.
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:
4165–4173.
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: 263–267.
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: 16213–16219.
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:
45713–45719.
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: 861–872.
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:
8283–8286.
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:
12430–12438.
Park S & Imlay JA (2003) High levels of intracellular cysteine
promote oxidative DNA damage by driving the Fenton
reaction. J Bacteriol 185: 1942–1950.
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: 3307–3309.
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:
47555–47563.
Rajagopalan KV (1997) Biosynthesis and processing of the
molybdenum cofactors. Biochem Soc Trans 25: 757–761.
FEMS Microbiol Rev 30 (2006) 825–840
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: 1031–1038.
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: 2277–2284.
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:
1699–1709.
Sauerwald A, Zhu W, Major TA, Roy H, Palioura S, Jahn D,
Whitman WB, Yates JR III, Ibba M & Söll D (2005) RNAdependent cysteine biosynthesis in Archaea. Science 307:
1969–1972.
Schmidt A & Jäger K (1992) Open questions about sulfur
metabolism in plants. Annu Rev Plant Physiol Plant Mol Biol
43: 325–349.
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:
11647–11657.
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: 3896–3903.
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 Cterminal domains of the NifU scaffold protein. Biochemistry
44: 12955–12969.
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:
583–593.
Steegborn C, Clausen T, Sondermann P, Jacob U, Worbs M,
Marinkovic S, Huber R & Wahl MC (1999) Kinetics and
inhibition of recombinant human cystathionine g-lyase.
Toward the rational control of transsulfuration. J Biol Chem
274: 12675–12684.
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: 21–31.
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:
1919–1928.
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: 28380–28383.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
840
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: 16555–16560.
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: 1659–1670.
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: 1564–1574.
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: 12210–12219.
Toohey JI (1989) Sulphane sulphur in biological systems: a
possible regulatory role. Biochem J 264: 625–632.
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 ironsulphur protein maturation. Nature 426: 172–176.
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: 8352–8358.
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: 59–71.
Wang R (2002) Two’s company, three’s a crowd: can H2S be the
third endogenous gaseous transmitter? FASEB J 16:
1792–1798.
Webb E, Claas K & Downs DM (1997) Characterization of thiI, a
new gene involved in thiazole biosynthesis in Salmonella
typhimurium. J Bacteriol 179: 4399–4402.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
D. Kessler
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: 1801–1809.
Wood JL (1987) Sulfane sulfur. Meth Enzymol 143: 25–29.
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: 14523–14532.
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:
8513–8518.
Zdych E, Peist R, Reidl J & Boos W (1995) MalYof Escherichia coli
is an enzyme with the activity of a bC-S lyase (cystathionase).
J Bacteriol 177: 5035–5039.
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:
1293–1302.
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:
2754–2758.
Zheng L, White RH, Cash VL & Dean DR (1994) Mechanism for
the desulfurization of L-cysteine catalyzed by the nifS gene
product. Biochemistry 33: 4714–4720.
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:
13264–13272.
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: 4562–4570.
FEMS Microbiol Rev 30 (2006) 825–840