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Journal of Experimental Botany, Vol. 55, No. 404,
Sulphur Metabolism in Plants Special Issue, pp. 1775–1783, August 2004
DOI: 10.1093/jxb/erh185 Advance Access publication 18 June, 2004
Plant adenosine 59-phosphosulphate reductase: the past,
the present, and the future
Stanislav Kopriva1,* and Anna Koprivova2
1
Albert-Ludwigs-University of Freiburg, Institute of Forest Botany and Tree Physiology,
Georges-Köhler-Allee 053, D-79110 Freiburg, Germany
2
Albert-Ludwigs-University of Freiburg, Plant Biotechnology, Schänzlestr. 1, D-79104 Freiburg, Germany
Received 26 January 2004; Accepted 27 April 2004
Abstract
The sulphate assimilation pathway provides reduced
sulphur for the synthesis of the amino acids cysteine
and methionine. These are the essential building blocks
of proteins and further sources of reduced sulphur for
the synthesis of coenzymes and various secondary
compounds. Several recent reports identified the adenosine 59-phosphosulphate reductase (APR) as the enzyme with the greatest control over the pathway. In this
review, a short historical excursion into the investigations of sulphate assimilation is given with emphasis on
the proposed alternative pathways to APR, via ‘bound
sulphite’ or via PAPS reductase. The evolutionary past
of APR is reviewed, based on phylogenetic analysis of
APR and PAPS reductase sequences. Furthermore,
recent biochemical analyses of APR that identified an
iron–sulphur centre as a cofactor, proposed functions
for different protein domains, and addressed the enzyme mechanism are summarized. Finally, questions
that have to be addressed in order to improve understanding of the molecular mechanism and regulation of
APR have been identified.
Key words: Adenosine phosphosulphate reductase, cysteine
biosynthesis, iron–sulphur centre, sulphate assimilation.
lipids. In plants, in addition, a variety of S-containing
secondary metabolites are synthesized, which often play an
important role in defence against pathogens and herbivores.
For incorporation into bio-organic compounds sulphate
has to be reduced in the pathway of assimilatory sulphate
reduction. This pathway is present in plants, algae, fungi,
and autotrophic prokaryotes, but is missing in Metazoa and
most parasitic prokaryotes and eukaryotes. Plant sulphate
assimilation is the major source of reduced sulphur for
animal and human diets. Nevertheless, the investigations
of sulphur metabolism were always fewer compared with
research on carbohydrates or nitrogen assimilation and, for
a long time, a consensual view on the pathway was missing
(Schmidt and Jäger, 1982). However, in the last few years
substantial progress in understanding plant sulphate assimilation has been achieved (Leustek and Saito, 1999; Leustek
et al., 2000; Hawkesford and Wray, 2000; Suter et al.,
2000; Höfgen et al., 2001). Several recent reports identified
adenosine 59-phosphosulphate reductase (APR) as the key
enzyme in this pathway and demonstrated many important
results on its biochemistry and regulation (Suter et al.,
2000; Bick et al., 2001; Kopriva et al., 2001). Therefore, in
this review, a short historical excursion into the investigations of sulphate assimilations is provided, with a focus on
adenosine 59-phosphosulphate reductase, its biochemical
characterization, and evolution, and finally questions on the
understanding of the molecular mechanism and regulation
of this enzyme are summarized.
Introduction
Historical overview
Sulphur is found in the amino acids cysteine and methionine that are essential components of peptides and proteins,
in iron–sulphur clusters and other cofactors, and as sulphonate group modifying proteins, polysaccharides, and
The sulphate assimilation pathway was first resolved in the
enteric bacteria Escherichia coli and Salmonella typhimurium using mutants auxotrophic for different sulphur
compounds (Jones-Mortimer, 1968; Kredich, 1971). To be
* To whom correspondence should be addressed. Fax: +49 761 2038302. E-mail: [email protected]
Journal of Experimental Botany, Vol. 55, No. 404, ª Society for Experimental Biology 2004; all rights reserved
1776 Kopriva and Koprivova
metabolized, the relatively inert sulphate must first
be activated. The initial activation step, an adenylation to
adenosine 59-phosphosulphate (APS) is catalysed by ATP
sulphurylase (ATPS). In the second step APS is further
phosphorylated by APS kinase to form adenosine 39phosphate 59-phosphosulphate (PAPS). PAPS is reduced
in a thioredoxin-dependent reaction by PAPS reductase
(CysH) to sulphite. In the second reduction step, sulphite
is reduced by a NADPH-dependent sulphite reductase to
sulphide. In bacteria, sulphide is finally incorporated into the
amino acid skeleton of O-acetyl-L-serine (OAS) forming
cysteine.
It has long been known that plants are able to take up
sulphate and convert it into cysteine (reviewed in Wilson,
1962). Sulphite and sulphide were identified as intermediates in sulphate reduction and the pathway was shown to
be stimulated by light (Fromageot and Perez-Milan, 1959;
Asahi, 1960). PAPS had been detected in plants (Schiff,
1959); therefore, in analogy with enteric bacteria, the same
sequence of reactions was proposed for plant sulphate
assimilation (Fig. 1). However, studies with the green alga
Chlorella revealed that APS rather than PAPS was reduced
(Tsang et al., 1971; Schmidt, 1972a). Among the products
of this reaction, sulphite bound onto a thiol carrier, which
could be replaced in vitro by glutathione (GSH), was most
prominent (Schiff and Hodson, 1970; Schmidt, 1972b). The
bound sulphite can be reduced further to bound sulphide
by a ferredoxin-dependent thiosulphonate reductase, and
react with OAS to form cysteine (Schmidt, 1973). The
enzyme catalysing the transfer of sulphate from APS to the
thiol carrier was named APS sulphotransferase (APSST)
(Schmidt, 1972b). APSST activity was detected in a variety
of plants and photosynthetic bacteria (Schmidt, 1975;
Schmidt and Trüper, 1977). Therefore, and because its
activity was highly regulated, APSST was considered to
represent the major sulphate-reducing enzyme in photosynthetic organisms, by contrast with enteric bacteria and yeast
which seemed to utilize PAPS for reduction (Brunold,
1990; Schmidt and Jäger, 1992). Nevertheless, a PAPS-
Fig. 1. Assimilatory sulphate reduction pathway in Escherichia coli.
dependent pathway of plant sulphate assimilation was never
excluded, especially when the purification of PAPS reductase from spinach had been reported (Schwenn, 1989).
In attempts to clone plant APS and/or PAPS reducing
enzyme(s) by complementation of E. coli mutants deficient
in cysH, three different cDNA clones were obtained from
Arabidopsis thaliana (Gutierrez-Marcos et al., 1996; Setya
et al., 1996). These cDNAs encoded isoforms of a novel
protein with three distinct domains: an N-terminal organelle
targeting peptide, a central part homologous to E. coli PAPS
reductase, and a C-terminal extension similar to thioredoxin. Since the enzyme produced sulphite from APS but
not from PAPS, it was named APS reductase (APR) (Setya
et al., 1996). In the following years several reviews tried to
establish the role of APR in sulphate assimilation, but came
to contradictory conclusions (Hell, 1997; Wray et al., 1998;
Leustek and Saito, 1999). Whereas Wray et al. (1998)
speculated that APS is directly reduced to free sulphite and
further to free sulphide by SiR, Leustek and Saito (1999)
revived the bound sulphite pathway, assuming that APR
acts as a sulphotransferase forming S-sulphoglutathione as
a reaction product. Sulphide may be formed in a reaction
catalysed by SiR from free sulphite resulting from a nonenzymatic reaction of S-sulphoglutathione with GSH.
The controversy about the enzyme catalysing the reduction of APS was resolved by Suter et al. (2000). The
APSST from Lemna minor was isolated and the corresponding cDNA was cloned. The deduced amino acid
sequence was 73% identical to that of APS reductase from
A. thaliana, revealing that APS sulphotransferase and
APS reductase were identical enzymes. This finding itself,
however, could not clarify the controversy about the enzyme name and reaction mechanism. The method of measuring APR activity (Brunold and Suter, 1990) cannot
distinguish between a free sulphite and sulphite bound to
a thiol as the reaction products. Thus, it is compatible
with both the free and the bound sulphite pathways and,
correspondingly, with both reductase and sulphotransferase
reaction mechanisms. From the published kinetic data
neither mechanism could be excluded (Schmidt, 1972b;
Abrams and Schiff, 1973; Schmidt and Jäger, 1992; Li
and Schiff, 1992). The analysis of reaction products of the
recombinant APR from L. minor, however, revealed that
free sulphite was the only reaction product detected under
non-oxidizing conditions, while S-sulphoglutathione was
only formed when oxidized glutathione was present in the
enzyme assay (Suter et al., 2000). This means that the
enzyme is a reductase and that the bound sulphite pathway
was most probably an artefact due to the presence of
oxidized thiols in the reaction assay. These conclusions
were corroborated by further biochemical characterization
of APR (Weber et al., 2000; Kopriva et al., 2001).
The major uncertainty in the pathway of sulphate
assimilation in plants was, therefore, the presence or absence of PAPS reductase. The Arabidopsis and rice genomes
Plant APS reductase
do not contain any genes homologous to E. coli PAPS
reductase, other than those encoding APR. However,
since such plant PAPS reductase may have a structure
completely divergent from that of the bacterial enzyme,
only analysis of plants lacking APR activity would prove
or exclude the PAPS-dependent sulphate assimilation in
plants. In higher plants, neither mutants nor transgenic
plants devoid of APR activity are known. For targeted
gene disruption, the moss Physcomitrella patens is an ideal
system because of high rates of homologous recombination
(Schaefer, 2002). To produce and analyse plants lacking
APR activity, the single copy gene encoding APR was
cloned from P. patens and disrupted (Koprivova et al.,
2002). This resulted in the complete loss of the correct
transcript and enzymatic activity. However, surprisingly,
the knockout plants were still able to grow on sulphate as
the sole sulphur source. Although PAPS reductase activity
could not be measured in the knockout plants, a cDNA and
gene coding for this enzyme were detected in P. patens. The
PAPS reductase from Physcomitrella does not contain the
thioredoxin-like domain, the two cysteine pairs binding the
FeS cofactor in APR are absent, and the protein seems to
contain a chloroplast targeting peptide (Koprivova et al.,
2002). The moss Physcomitrella patens is the first plant
species where PAPS reductase was confirmed on the molecular level and also the first organism where both APS
and PAPS-dependent sulphate assimilation co-exist.
Figure 2 shows the new, now generally accepted, scheme
of the sulphate assimilation pathway in plants. Sulphate is
adenylated by ATP sulphurylase to APS, which is reduced
by APS reductase to free sulphite; the electrons are derived
from glutathione. APS can also be further phosphorylated
to PAPS, which serves as a source of activated sulphate for
a variety of sulphotransferases. At least in some lower
plants PAPS can be directly reduced to sulphite by PAPS
reductase. The significance of this reaction in higher plants
is uncertain, which is demonstrated by a dashed line in the
Fig. 2. Assimilatory sulphate reduction pathway in higher plants, current
view.
1777
scheme. Sulphite is reduced by a ferredoxin-dependent
sulphite reductase to sulphide and incorporated into the
amino acid skeleton of OAS to form cysteine.
Biochemical characterization of APR
APR catalyses a thiol-dependent two-electron reduction
of APS to sulphite (Suter et al., 2000). APR activity is
measured as acid volatile radioactivity after incubation
with [35S]APS and thiols. The sulphite evolved after the
addition of H2SO4 is trapped in triethanolamine and the
radioactivity is determined in the scintillation counter
(Brunold and Suter, 1990). The reaction has a pH optimum
of 8.5–9, is stimulated by concentrations of sulphate higher
than 0.6 M, and is inhibited by 59-AMP (Brunold and Suter,
1990; Setya et al., 1996). The enzyme was first purified
from the green alga Chlorella as a protein of molecular
weight greater than 300 kDa (Schmidt, 1972b). However,
because of its limited amount in cells it has not often been
studied in detail. Li and Schiff (1991) extracted APR from
Euglena gracilis and purified to homogeneity a tetramer
of 25 kDa subunits held together by disulphide bonds. The
enzymes from Chlorella and Euglena differed not only in
their molecular weights, but also in the efficiency of APS
reduction using different thiol compounds as electron
donors. While APR from Chlorella utilized GSH and
dithiothreitol (DTT) with the same efficiency, the activity
of Euglena APR with GSH reached only 23% of the
activity with DTT (Schmidt, 1972b; Li and Schiff, 1991). A
further source for APR purification was the marine macroalga Porphyra yezoensis where the subunit Mr was estimated to be 43 000 (Kanno et al., 1996). The molecular
weight calculated from the deduced amino acid sequence of
the APR cDNAs from Arabidopsis and Lemna was also
43 000 (Gutierrez-Marcos et al., 1996; Setya et al., 1996;
Suter et al., 2000). The molecular mass of native Lemna
APR (Suter et al., 2000) and recombinant APR2 from A.
thaliana (M Weber and S Kopriva, unpublished data) was
estimated to be 91 kDa, revealing that, in higher plants, the
enzyme is a dimer of 43 kDa subunits. The APR monomers seem to be joined by a disulphide bond, because after
treatment with thiols APR elutes from the column as
monomer. This disulphide is most probably formed by
the only Cys conserved among APS and PAPS reductases
(Cys248 of the ECGLH motif in APR2), since the molecular
weight of modified APR where this Cys was exchanged
with Ser also corresponded with that of an APR monomer
(M Weber and S Kopriva, unpublished results). Accordingly, after pre-incubation with thiols (DTT, GSH) in the
absence of APS or AMP, APR loses its activity (S Kopriva,
unpublished results; Bick et al., 2001). On the other hand,
APR expressed in E. coli lacking a thioredoxin reductase
(trxB) was 11–45-fold more active than the enzyme expressed in the wild-type strain (Bick et al., 2001). Only the
APR1 isoform of A. thaliana, but not APR2 and APR3,
1778 Kopriva and Koprivova
was activated in the trxB strain, revealing possible isoformspecific differences. Bick et al. (2001) concluded from
their data that a regulatory Cys pair is present in APR1, which
enables rapid post-translational regulation of APR activity
in vivo. However, a change between an active dimer and
inactive monomer might be an alternative explanation.
Localization of APR
Sulphate reduction was demonstrated in isolated chloroplasts (Schmidt and Trebst, 1969) and, correspondingly, in
spinach the APR activity was localized in chloroplasts
(Schmidt, 1976; Fankhauser and Brunold, 1978). Western
analysis of pea chloroplast fractions detected APR protein
in the stroma, but not in any of the membrane fractions
(Prior et al., 1999) and immunogold labelling revealed
APR in chloroplasts of three Flaveria species with different
types of photosynthesis (Koprivova et al., 2001). The comparison of the N-terminal sequence of purified APR from
Lemna with the amino acid sequence deduced from the
cDNA confirmed the presence of a chloroplast targeting peptide encoded by the full-length cDNA (Suter et al.,
2000). Indeed, recombinant APR from Catharanthus
roseus could be imported into intact pea chloroplasts and
correctly processed there (Prior et al., 1999).
APR is composed from two distinct domains
The mature APR consists of two distinct domains, a PAPS
reductase-like one and a thioredoxin-like one. When a
truncated APR lacking the thioredoxin-like domain was
expressed in E. coli and purified, low or no APR activity
was measured (Bick et al., 1998; Prior et al., 1999; Weber
et al., 2000). Adding the corresponding, separately expressed, thioredoxin-like domain reconstituted APR activity (Bick et al., 1998; Prior et al., 1999). The activity could
be recovered after the addition of thioredoxin (Prior et al.,
1999; Weber et al., 2000). Only thioredoxin m, but not
thioredoxin f, was able to reconstitute the activity of the A.
thaliana APR2 isoform (Weber et al., 2000). The CXXC
thioredoxin active site motif found in the C-terminal
domain of APR, however, also occurs in glutaredoxin,
another class of thiol:disulphide oxidoreductases. Glutaredoxin does not require an enzymatic reduction, like
thioredoxin by thioredoxin reductase, but is reduced by
glutathione, whose oxidized form must, in turn, be reduced
by glutathione reductase (Rietsch and Beckwith, 1998).
Glutaredoxin was identified as an alternative electron donor
for sulphate reduction in an E. coli mutant lacking thioredoxin (Tsang and Schiff, 1978). Glutaredoxin, but not
thioredoxin, also catalyses the reduction of dehydroascorbate (Washburn and Wells, 1999). Although the sequence
of the C-terminal domain of APR is more similar to
thioredoxin than to glutaredoxin, it possesses the enzymatic
activity of glutaredoxin, since (i) glutathione is an efficient
hydrogen donor for APR in vitro (KM=0.6–3 mM)
(Schmidt, 1972b; Bick et al., 1998; Prior et al., 1999),
(ii) APR and the C-terminal domain catalyse the glutathione-dependent reduction of dehydroascorbate and hydroxyethyldisulphide and possesses ribonucleotide reductase
and protein disulphide isomerase activities (Bick et al.,
1998; Prior et al., 1999), (iii) the C-terminal domain can
complement E. coli mutants lacking glutaredoxin, but not
those defective in thioredoxin (Bick et al., 1998), and (iv)
in the C-terminal domain of APR from the moss Physcomitrella patens the CXXC motif is changed to CXXS, an
alteration tolerated by glutaredoxin but not thioredoxin
(Koprivova et al., 2002).
The two domains of APR can not only be expressed
separately and possess independent enzyme activities; they
also seem to catalyse distinct steps in APS reduction. The
first approach to study the molecular mechanism of the
APR reaction led to the identification of a reaction intermediate, with sulphite covalently bound to a cysteine
residue conserved between APS and PAPS reductases
(Weber et al., 2000). The [35S]sulphite was lost from the
protein by reaction with reduced thiols or sulphite, but also
by incubation at 37 8C without the addition of thiols. As
truncated APR2, missing the C-terminal domain, remained
labelled at 37 8C, the thioredoxin-like domain seems to
be required to release the bound SO2ÿ
3 from the reaction
intermediate. In analogy with the S-sulphoglutathione
discussion, it has to be noted that the reaction intermediate
was formed even in conditions where APR was inactive
(pH 6) and, thus, it could not result from a chemical
reaction between free sulphite and an enzyme dimer
(Weber et al., 2000). The APR reaction can thus be divided
into three independent steps: a transfer of sulphate from
APS to the active cysteine residue, the release of the
sulphite by the C-terminal domain, probably by forming
a intramolecular disulphide bridge, and recovery of the
active enzyme dimer by reaction with thiols (Fig. 3). For the
first step, which results in binding of sulphite to APR,
neither the thioredoxin-like domain nor an external electron
source is required and also the electrons necessary for
the sulphite release are provided by the APR protein. The
molecular mechanisms of these reaction steps are, however, far from being understood. The identification of this
reaction intermediate thus corroborated the reductase mechanism of APR. Similar, but not identical, intermediates as
found in the APR reaction, with sulphite covalently bound
to a protein were described previously (Abrams and Schiff,
1973; Schmidt, 1973; Li and Schiff, 1992). Schmidt (1973)
and Abrams and Schiff (1973) obtained a low molecular
weight protein with bound sulphite in Chlorella extracts
only in the presence of thiols, giving the bound sulphite
pathway its name. In addition, APR from Euglena gracilis
was shown to form a stable reaction intermediate. The
enzyme, however, was not characterized further; but it
differs in composition from the higher plant APR (Li and
Schiff, 1992). By contrast, the reaction mechanism of yeast
Plant APS reductase
1779
Fig. 3. Reaction steps in APS reduction by APR. The squares and circles represent the N-terminal and C-terminal domains, respectively. The active site
cysteine residue, as well as the Cys possibly forming an intramolecular disulphide, are indicated by S.
Fig. 4. Neighbor–Joining tree of APR and PAPS reductase protein
sequences. The sequences were retrieved from GenBank, aligned with
Clustal W, and the tree was constructed with the MEGA 2.1 software.
The accession numbers for the protein sequences used are as follows
(from the top of the tree): T49106, P92979, P92981, AAB05871,
AAF18999, CAB65911, AY594279, CAD22096, AAM18118, T52251,
O05927, AAF23174, AAR35093, CAD16130, BX640413 (DNA sequence), P56891, AAK86625, D90470, AAL64292, P71752,
CAC33945, CAD75117, AAO05823, P94498, AAF28889, P18408,
CAE76190, CAD32963, AY594280, AAP99141, CAE08679, P72794,
Q8P607, P57501, P17853, P17854. The horizontal line divides the tree in
the APR clade and the PAPS reductase clade. Asterisk marks the
bispecific sulphonucleotide reductase from Bacillus subtilis (Berndt
et al., 2004).
PAPS reductase has the following sequence of reactions: an
interaction of the enzyme with reduced thioredoxin, storage
of two electrons probably at the conserved Cys residues of
the enzyme dimer, interaction with PAPS, and release of
sulphite and adenosine 39,59-bisphosphate (Schwenn et al.,
1988; Berendt et al., 1995).
APR contains a FeS cluster
APRs from A. thaliana and C. roseus which had been
expressed in E. coli, were described as proteins without
prosthetic groups or cofactors (Gutierrez-Marcos et al.,
1996; Setya et al., 1996; Prior et al., 1999). On the other
hand, APR was purified from L. minor as a yellow-brown
protein (Suter et al., 2000) indicating the presence of
a cofactor, possibly FAD or/and an FeS cluster. UV/visible
spectra of recombinant APR from Lemna and all three
isoforms from A. thaliana indicated the presence of an
iron–sulphur centre and, indeed, iron and acid-labile
sulphide were found to bind to the APR protein (Kopriva
et al., 2001). Electron paramagnetic resonance and Mössbauer spectroscopy confirmed the presence of a diamagnetic
[4Fe–4S]2+ cluster. This cluster is unusual since only three
of the iron sites exhibited the same Mössbauer parameters,
one of these being either tetragonally FeS3X co-ordinated,
with X representing a non-sulphur ligand (C, N, O), or
trigonally sulphur-co-ordinated. There is no signature for
binding of an FeS cluster in the sequence of plant APR.
However, the major difference between the N-terminal
part of APR and PAPS reductases is the presence of two
additional cysteine pairs in the plant enzyme. Only three
from these four additional cysteine residues can bind the
FeS cluster, thus explaining its extraordinary characteristics
(Kopriva et al., 2001). It has to be noted that dissimilatory
APS reductase, an enzyme catalysing the reduction of APS
in anaerobic, sulphate-reducing bacteria such as Desulphovibrio sp., also carries two [4Fe–4S] centres in addition to
FAD. The structure of dissimilatory APR is, however,
completely different from that of plant APR. The protein is
a dimer of a large FAD-containing subunit with a small one
possessing two [4Fe–4S] clusters (Verhagen et al., 1994);
there is no sequence homology among these proteins. Thus,
plant APR represents a novel type of APS reductase with
a [4Fe–4S] centre as the sole cofactor.
Bacterial assimilatory APR
Recently, another type of APS reductase was characterized
in several bacteria. Although, as discussed above, it was
long believed that heterotrophic bacteria reduce sulphate
via PAPS, an APS-dependent sulphate assimilation was
confirmed in several species of Rhizobiaceae, Pseudomonas, Burkholderia, Ralstonia, Mycobacterium, and Bacillus (Abola et al., 1999; Bick et al., 2000; Kopriva et al.,
2002; Williams et al., 2002). The corresponding enzymes,
encoded by CysH genes, are more similar to the N-terminal
part of plant APR than to PAPS reductase of E. coli and
are lacking the thioredoxin-like domain. Accordingly,
the APR activity of these proteins is highly stimulated by
the addition of thioredoxin. The bacterial assimilatory APR
1780 Kopriva and Koprivova
also possesses the two Cys pairs conserved with the plant
enzyme which are not found in PAPS reductase from
E. coli. As these Cys residues participate in binding of the
iron–sulphur cluster in plant APR (Kopriva et al., 2001), it
is not surprising that the APRs from Sinorhizobium meliloti
and Pseudomonas aeruginosa bind an FeS centre with
the same characteristics as the FeS cluster in plant APR
(Kopriva et al., 2002). Very recently, the CysH gene
product from Bacillus subtilis was characterized as a bifunctional APS/PAPS reductase (Berndt et al., 2004). This
protein has higher affinity for PAPS than for APS and
requires thioredoxin or glutaredoxin for catalytic activity.
Most importantly, this sulphonucleotide reductase also
binds the FeS cofactor, which is rapidly lost upon exposure
to air. After chemical removal of the cofactor APS reduction was not detectable, whereas residual PAPS reductase activity could still be measured (Berndt et al.,
2004). Reduction of APS versus PAPS thus seems to be
dependent on the FeS cluster and the two conserved Cys
pairs seem to represent a marker to distinguish CysH
homologues capable of APS reduction. The ability of the B.
subtilis APS/PAPS reductase to react with both sulphonucleotides seems to be an exception, since the recombinant
APRs from P. aeruginosa and S. meliloti were clearly
monospecific for APS (Abola et al., 1999; Bick et al., 2000;
Kopriva et al., 2002). If any of the other bacterial
assimilatory APRs are bispecific like the B. subtilis enzyme, what structural features are the determinants of this
bispecificity, and which sulphonucleotide is actually reduced in vivo remains to be elucidated.
Evolution of APR
The evolutionary past of APR is anything but straightforward, especially after cloning a PAPS reductase from the
moss P. patens (Koprivova et al., 2002). The plant APR
gene most probably originated from a fusion between
prokaryotic genes for APS or PAPS reductase and thioredoxin. Since all APRs isolated or cloned from higher and
lower plants, and the APR from the green algae Chlamydomonas rheinhardtii (Ravina et al., 2002) and Enteromorpha intestinalis (Gao et al., 2000), have the same structure, this fusion must have occurred early in the evolution
of plants. However, the origin of the prokaryotic (P)APS
reductase gene involved in this fusion, the existence of
at least four different enzymes catalysing reduction of
activated sulphate (plant APR, assimilatory bacterial APR,
dissimilatory APR, and PAPS reductase), and the distribution of these enzymes in different organisms are not clear,
yet. Sulphate assimilation is present in plants, algae, fungi,
and many autotrophic prokaryotes. Among the 123 species
of the Archae and Eubacteria superkingdoms, whose
genomes have been completely sequenced to date, only
64 possess homologues of APR or PAPS reductase. Among
these 64 species, an assimilatory APR (including the
bispecific APS/PAPS reductase from B. subtilis) is present
in 38 species whereas PAPS reductase can be found in
26 species, when the presence of the two Cys pairs serves
as a marker to distinguish between these two proteins
(Kopriva et al., 2002; see above). Utilization of APS for
sulphate reduction is thus much more widely distributed
than it was originally believed.
During the search of sequence databases for homologues
of plant APR and PAPS reductase a putative PAPS reductase was identified in the EST collection from the spike
moss Selaginella lepidophylla, a vascular plant (S Kopriva,
unpublished results). This is of great importance because it
proves that the PAPS reductase found in P. patens (Koprivova et al., 2002) did not originate from a late horizontal
gene transfer to Bryophytes, but must have been present in
the common ancestor of Bryophytes and vascular plants.
The APR amino acid sequences from different plant and
algae species are well conserved, containing 60–80%
identical amino acid residues. The PAPS reductase-like
domains of plant APR are 22–27% identical to the PAPS
reductase from E. coli or yeast and 40–50% to the assimilatory bacterial APR. Figure 4 shows a Neighbor–Joining
tree constructed from representative APR and PAPS
reductase sequences, retrieved from the GenBank. As
described in Kopriva et al. (2002), using a larger but less
diverse set of sequences, the tree is divided into two major
clades. The first clade contains plant and algae APRs,
together with many putative and confirmed assimilatory
bacterial APRs and the bispecific B. subtilis enzyme, the
other clade comprises PAPS reductases from the two plant
species, fungi, enteric bacteria, and cyanobacteria. In all
organisms of the ‘APR’ clade tested to date, APR activity
was detected in protein extracts or in corresponding recombinant proteins, whereas the organisms of the ‘PAPS
reductase’ clade seem to possess the PAPS reductase
activity exclusively (Abola et al., 1999; Bick et al., 2000;
Kopriva et al., 2002; Williams et al., 2002; Berndt et al.,
2004). In a reciprocal approach, a partial APR gene was
cloned from a cyanobacteria Plectonema 73110, which, in
contrast to other cyanobacteria, possess an APS reducing enzyme (Schmidt, 1977) that clustered with the APS
reductases from plants and several c-proteobacteria
(Kopriva et al., 2002). As expected from the discussion
above, all proteins from the ‘APR’ clade possess the two Cys
pairs, which are not present in the enzymes of the ‘PAPS
reductase’ clade.
Although previously considered to be the major bacterial
enzyme for the reduction of activated sulphate, PAPS
reductase seems to be restricted to only few groups of cproteobacteria and cyanobacteria. As APS reducing species
are found also in these phyla and since the APR tree
topology (Fig. 4) does not correspond to the evolutionary
relationship based on rRNA, a horizontal gene transfer
must have played an important role in today’s distribution
of the two enzyme activities. This is interesting, because
Plant APS reductase
also the evolution of dissimilatory APS reductase was
affected by frequent horizontal gene transfers (Friedrich,
2002). From the two enzymes, APR seems to be evolutionarily older than PAPS reductase since (i) the PAPS
reductase only occurs in two Eubacterial phyla, whereas
APR was confirmed in most taxonomical groups of
Eubacteria, (ii) dissimilatory sulphate-reducing bacteria
and Archae possess APS reductase which contains the FeS
cluster as a cofactor; and (iii) the activation to APS requires
less energy than to PAPS (see discussion in Kopriva et al.,
2002). The evolution of PAPS reductase, which does not
need the iron–sulphur cluster, might have been an adaptation to an iron- and/or sulphur-poor environment.
Open questions and future research needs
Although much progress in the characterization of APR has
been made in recent years, there are still many important
questions to answer. To date, the exact reaction mechanism
and the role of the FeS cofactor in catalysis are not known.
For this purpose, a crystallization of the plant enzyme and
a structure analysis would certainly deliver the necessary
data. The evolutionary studies of APR and PAPS reductase
would profit from an extended analysis of the distribution
of these enzymes in lower plants, algae, and primitive
Eukaryotes. Further characterization of the bifunctional B.
subtilis enzyme will be very important for understanding
the evolution of the two enzymes in bacteria. Although
APR is highly regulated in plants (for a review see Leustek
et al., 2000; Brunold et al., 2003; Kopriva and Koprivova,
2003), no immediate signals and transcription factors
responsible for this regulation are known. Therefore,
molecular analysis of APR regulation in plants and the
biochemical analysis of the role of APR in the control
of sulphate assimilation would be most important. Here,
a genetic approach promises the most efficient way to
address the APR regulation at the molecular level. Studying
the latter question Vauclare et al. (2002) revealed that APR
possesses a very high control over the flux through sulphate
assimilation. APR would, therefore, be a good target for
genetic engineering in order to obtain plants with a higher
content of reduced sulphur for nutrition or increased stress
tolerance. Indeed, it seems that increased APR activity
results in greater flux through the pathway. Tsakraklides
et al. (2002) showed that overexpression of APR in
Arabidopsis led to higher contents of reduced sulphur
compounds. The inorganic compounds sulphite and thiosulphate were increased to a greater extent than the thiols
cysteine and glutathione. These results revealed that, for the
synthesis of thiols, the availability of organic acceptors is as
important as the production of sulphide by the sulphate
assimilation pathway (Tsakraklides et al., 2002). Therefore,
more extended flux control analysis using plant material
with increased APR activity is needed to evaluate the contribution of sulphate reduction and the availability of
1781
carbohydrates in the control of cysteine synthesis and
to suggest the most efficient way to produce plants with
a maximal capacity for the synthesis of thiols. Thus,
considering the central role of APR in sulphate assimilation
and in the primary metabolism of plants, more about this
interesting enzyme will surely be heard in future and the
open questions will hopefully move from substantial ones
to comparatively fine details.
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