240

Plant — the reduction of to sulfite Julie Ann Bick and Thomas Leustek∗

Until recently the pathway by which reduce activated matter of contention and is the focus of this review. sulfate to sulfite was unresolved. Recent findings on two Skipping this step for the moment, the remaining reactions termed 5′-adenylylsulfate (APS) sulfotransferase and in the pathway to include the reduction of APS reductase have provided new information on this topic. sulfite to sulfide catalyzed by -dependent sulfite On the basis of their similarities it is now proposed that these reductase [6], then assimilation of inorganic sulfide by are the same . These discoveries confirm that the sulfhydration of O-acetylserine [7••]. The second the sulfate assimilation pathway in plants differs from that in assimilation pathway branching from APS is used for the other sulfate assimilating organisms. synthesis of a variety of sulfated compounds carried out by specific sulfotransferases (ST’s). These enzymes use the phosphorylated derivative of APS, 3-phosphoadenosine- Addresses 5′-phosphosulfate (PAPS)[4••,5••], formed by APS kinase Biotechnology Center for Agriculture and the Environment, Rutgers University, 59 Dudley Road, Foran Hall, New Brunswick, New Jersey (AK). 08901-8520 USA ∗e-mail: [email protected] This paper focuses on the latest information on the Current Opinion in Biology 1998, 1:240–244 enzyme catalyzing the reduction of APS. The recent purification of APSSTase from a marine alga provides the http://biomednet.com/elecref/1369526600100240 first evidence on the catalytic properties of this enzyme. At  Current Biology Ltd ISSN 1369-5266 the same time, cDNAs have been cloned from the higher Abbreviations plant Arabidopsis thaliana that encode an enzyme termed AK 5′-adenylyl sulfate kinase APS reductase that shows similarities to APSSTase. We APR 5′-adenylylsulfate reductase propose that they are the same enzyme and discuss their APS 5′-adenylylsulfate properties in relationship to a role in sulfate assimilation. APSSTase 5′-adenylylsulfate sulfotransferase PAPS 3′-phosphoadenosine-5′-phosphosulfate

Figure 1 Introduction NADP+ This article focuses on a single aspect of a broad subject NADPH H+ area. It is prompted by the recent findings concerning one of the primary steps in the sulfate assimilation GR O-acetylserine GSSG 2– pathway of higher plants. Readers who are interested in GSH 2– Cysteine SO3 S a comprehensive treatment of plant are • • APR SiR OASTL referred to several recent reviews [1 –3 ]. 2– (APSSTase) SO4 APS Higher plants are dependent on inorganic sulfate to fulfil AS their nutritional sulfur requirement. Sulfate is a major AK PAPS Sulfated compounds anionic solute and is required for the synthesis of many ST's different metabolites and coenzymes. After uptake from Current Opinion in Plant Biology the soil, sulfate is either accumulated and stored in the or it is incorporated into organic compounds The sulfate assimilation pathway in plants. Sulfate is activated by in a process referred to as assimilation. Sulfur can be ATP sulfurylase (AS) forming 5′-adenylylsulfate (APS). APS is a assimilated in one of two ways — it is either incorporated branch point intermediate that is used for the reduction pathway •• •• leading to formation of cysteine (upper pathway) or the sulfation as sulfate in a reaction termed sulfation [4 ,5 ], or it is pathway leading to synthesis of various sulfated compounds (lower first reduced to sulfide, the substrate for cysteine synthesis. pathway). The sulfation pathway begins by phosphorylation of APS In plants the majority of sulfur is assimilated in the to 3′-phosphoadenosine-5′phosphosulfate (PAPS) by APS kinase reduced form. (AK). PAPS is the substrate for a variety of sulfotransferases (ST’s). In the cysteine pathway, the first reduction step is the topic of this article. We argue that the enzyme APS reductase (APR) is identical The plant sulfate assimilation pathways are depicted to APS sulfotransferase (APSSTase), a -dependent in Figure 1. The prerequisite step for all metabolic reductase. Its activity is driven by reduced glutathione (GSH) which fates of sulfate is its activation by linkage to 5′-AMP, is recycled from oxidised glutathione (GSSG) through the action forming 5′-adenylylsulfate (APS), a reaction catalyzed of NADPH-dependent (GR). The subsequent by ATP sulfurylase. APS lies at a metabolic branch reactions are the reduction of sulfite to sulfide by sulfite reductase (SiR) and assimilation of sulfide into cysteine by O-acetylserine () point — it is used either for sulfation or for the reduction (OASTL). pathway. It is the first reduction step that has been a Plant sulfur metabolism — the reduction of sulfate to sulfite Bick and Leustek 241

Sulfate reduction in plants analogs) stabilize the enzyme against these inactivating The long-held dogma concerning sulfate reduction stated treatments. that, in plants, APS is the substrate for this reaction and the enzyme APS sulfotransferase (APSSTase) [8]. Although the reaction mechanism of APSSTase has not For a number of reasons, however, the APS pathway been addressed, this is a central issue that should be in plants was not universally accepted [9] — despite explored with the purified enzyme. As already mentioned years of research, definitive evidence for the existence APSSTase was proposed to be a sulfotransferase. For of APSSTase was not forthcoming. One report of its example, with glutathione as the acceptor S-sulfoglu- purification from Euglena gracilis was confounded by the tathione is formed. It was acknowledged, however, that unreasonably low specific activity of the pure enzyme this hypothesis is formed on the basis of inconclusive [10]. Subsequently, another group reported that, in vitro, data and that it is equally plausible that the enzyme is plant APS kinase displays APS sulfotransferase activity a reductase that forms free sulfite [8]. Due to its high as a side reaction [11]. This result, combined with reactivity sulfite can readily form a thiosulfonate with several physical similarities, prompted the idea that reduced [23], possibly explaining the presence of perhaps APS sulfotransferase is a kinetic artefact [11]. thiosulfonate in APSSTase reactions. In contrast to this uncertainty, it is widely believed that prokaryotes (including ) and fungi use PAPS The purification of APSSTase from a macroalga is the for sulfate reduction via the enzyme PAPS reductase first direct and convincing evidence for an APS-dependent [12,13]. Recently, however, direct evidence was published reduction pathway in plants. Additional and complemen- by three independent groups. confirming the existence of tary evidence for such a pathway was contemporaneously an APS-dependent pathway in plants [14••–16••]. obtained using molecular genetic methods as described in the next section.

APS sulfotransferase The APR cDNAs encoding APS reductase Although APSSTase has long been the focus of inves- APS reductase (APR) was identified serendipitously by searching for cDNAs encoding plant PAPS reductase tigation [17] its purification to homogeneity proved to •• •• be an elusive goal. Kanno et al. [14••] finally succeeded [15 ,16 ]. Three different cDNAs were cloned from in isolating the enzyme from a marine macroalga by Arabidopsis thaliana (APR1, 2 and 3) that are able to maintaining high levels of ammonium sulfate throughout complement the cysteine auxotrophy of an the purification procedure. Previous publications noted the cysH (PAPS reductase) mutant strain. The APR cDNAs lability of APSSTase and that it could be stabilized with encode individual members of a small, highly conserved high concentrations of sulfate salts [18], but this finding gene family. was never incorporated into a purification scheme. Analysis of the APR isoenzymes revealed that, unlike PAPS reductase, they use APS preferentially over PAPS. As reflected by its name, APSSTase was proposed to This property was demonstrated both by in vitro assays catalyze the transfer of sulfate from APS to a thiol acceptor and by in vivo functional complementation of a cysC molecule forming a thiosulfonate [8]. In vitro, many dif- (APS kinase) mutant of E. coli with the APR cDNAS. ferent reduced thiol compounds, including dithiothreitol, An APS reductase would be expected to bypass both can serve as the acceptor. The physiological acceptor was the cysC and cysH mutations in E. coli. Another key envisaged to be glutathione, primarily because it is an difference is that, while PAPS reductase is dependent efficient substrate and is the most abundant thiol in the on the proteins or glutaredoxin as a stroma in which APSSTase is localized [19]. source of electrons, the activity of the APR enzymes is not The algal enzyme shows a kinetic constant for glutathione stimulated in vitro by the addition of E. coli or Spirullina sp. of 0.6 mM, a value that is well below the physiological thioredoxin. Also, the APR cDNAs are able to complement concentration in the stroma, reported to be between 3 and a thioredoxin/glutaredoxin double mutant of E. coli. 10 mM [20,21]. This is a key finding that supports much of the earlier work on this enzyme. For example, it has long The properties of the APR enzymes are remarkably similar been known that sulfite production from sulfate is not light to those of APSSTase with respect to optimum assay dependent, requiring only ATP and glutathione (it is not conditions, kinetic constants and inhibitors. A key feature influenced by NADPH or ferredoxin) [22]. is that like APSSTase the APR enzymes are able to use a variety of reduced thiol compounds such as dithiothreitol Purified algal APSSTase has a high catalytic activity and glutathione as a sole source of electrons. The enzyme –1 –1 (10.3 µmol min mg ) and a high affinity for APS shows a Km for glutathione of 0.6 mM and can only (Km = 2.1 µM). It shows a high pH optimum (9.0–9.5) complement the cysH mutation of E. coli if the strain and it is maximally active in the presence of high produces glutathione (Bick and Leustek, unpublished sulfate concentrations (0.5 M). The enzyme is sensitive to data). Although the APR enzymes have not yet been inactivation by heat and dithiothreitol, but not glutathione. directly linked with APSSTase the similarity of their High concentrations of sulfate salts and APS (or APS catalytic features is a strong argument that they are the 242 and metabolism

same enzyme. The derived sequences of the Evolution of APR enzymes APR cDNAs may reveal how APS reductase (and probably Despite the difference in substrate specificity the APR APSSTase) might function with only glutathione as a enzymes are clearly related in sequence to PAPS reductase reductant. The APR enzymes are composed of three from prokaryotes and fungi, but they are not related to domains (Figure 2). At the amino terminus is a region that APS reductase from dissimilatory sulfate reducing ; resembles a transit ; this would be cleaved from the this enzyme is a heteromeric iron-sulfur flavoprotein [27]. protein following import into the chloroplast. Adjacent to To avoid confusion, we propose that the plant enzyme this is the amino-terminal domain of the mature protein be referred to as the plant-type APS reductase. A recent that is homologous with PAPS reductases from a variety search of the DNA sequence databases revealed that of sulfate assimilating organisms. At the carboxyl end PAPS reductase from Pseudomonas aeruginosa (GenBank is a domain that resembles thioredoxin. The fusion of accession #U95379) and Rhizobium tropici (AJ001223) has reductase and into a single protein suggests that the highest homology with the reductase domain of the the thioredoxin-like domain may serve as an exclusive APR enzymes (∼70% amino acid similarity), far greater electron donor for the reductase domain. But what about than that with PAPS reductase from enteric bacteria, the interaction with glutathione? cyanobacteria or fungi (with which the reductase domain shares only ∼50% amino acid similarity).

Figure 2 The evolution of the APR gene family in A. thaliana was explored by analysis of its genomic sequences [28•]. Figure 3 shows that APR1 is encoded by three exons that are separated by two introns. In contrast, APR2 and APR3 are composed of four exons that are separated by three TP Reductase domain trx/grx domain introns: the salient difference being that APR1 lacks the Current Opinion in Plant Biology intron that separates exons 2 and 3 in APR2 and APR3. At the level of nucleotide homology, the coding sequence of Domain structure of the APR enzymes. The APR enzymes are APR1 is more closely related to APR3 (78% identity) than composed of three domains. The amino terminal domain is a to APR2 (68% identity). This suggests that APR1 emerged transit peptide (TP) required for localization to and is proteolytically removed to form the mature enzyme. The mature by duplication of an ancestral APR1/APR3 gene followed enzyme is composed of a reductase domain (shaded dark) by the loss of exon 2. The only PAPS reductase gene that homologous with bacterial PAPS reductase, and a carboxyl terminal is known to be interrupted is from the filamentous domain (shaded light) homologous with thioredoxin (trx). Emericella nidulans (GenBank accession #X82555). This gene contains a single intron that interrupts the coding sequence at a position very close to the position of intron Thioredoxin and related proteins are redox-active proteins 2 in APR1 and intron 3 in APR2 and APR3. that function with a number of different reductases [24,25]. A hallmark of these proteins is an A question of key significance in the evolution of consisting of two cysteine residues, spaced two amino the APR enzymes is how three distinct domains — the acids apart, that switch between the dithiol and disulfide. transit peptide, the reductase domain, and the Plants contain thioredoxin proteins in both the thioredoxin/glutaredoxin domain — became fused into a and chloroplast and these are reduced by NADPH-depen- single polypeptide. One popular idea on gene evolution dent or ferredoxin-dependent thioredoxin reductases. The is that the domains of proteins arise through the accretion closely related protein, glutaredoxin, is reduced by glu- of separate exons [29]. If this were the case in APR tathione, which is itself reduced by NADPH-dependent evolution one might expect to find an intron separating glutathione reductase. The natural redox systems used the exons encoding individual domains. This appears by these proteins are highly specific. For example, in to be the case with respect to the transit peptide. In contrast to glutaredoxin, thioredoxin is unable to accept all three genes it is encoded by exon 1 although the electrons from glutathione. Interestingly, although the protease cleavage site is encoded at the beginning of exon carboxyl domain sequence shows greater overall homology 2. In contrast, the other domains are not separated by with thioredoxin, the amino acid residues between the an intron, rather a portion of the reductase domain and active site are more typical of glutaredoxin. It the thioredoxin/glutaredoxin domain are fused into single has been demonstrated that these amino acids influence exon. This finding does not help to clarify the evolutionary the redox potential of the cysteines and, therefore, the origin of the gene. An interesting point, however, is that redox system that the proteins can react with [26]. Indeed, in APR1 there is a nearly perfect 12 basepair sequence we have shown that the carboxyl domain functions more duplication located at the 5′ end of intron 2 (gtatatttcgat) like glutaredoxin than thioredoxin. The implication of this and another within exon 3 (gtatatatcgat) at a position result is that sulfate reduction in plants may ultimately be that is approximately at the border between the reductase driven by NADPH through the activity of a glutathione and thioredoxin/glutaredoxin-encoding sequences. Similar, reductase as depicted in Figure 1. but less well conserved sequences, are present at the Plant sulfur metabolism — the reduction of sulfate to sulfite Bick and Leustek 243

may provide a basis for regulation of enzyme level, namely Figure 3 that changes in alter the protein level.

Conclusions The next few years promise to be an exciting time APR1 for the community of plant sulfur researchers. Gene cloning has emerged as a powerful tool to define the genes and enzymes of the pathways APR3 in plants and the effort of cataloging genes continued in 1997 [33•,34••,35••36•]. The full potential of molecular methods, however, has yet to be realized. We expect that APR2 soon the biochemistry, enzymology and cell biology of the sulfur assimilation pathway will be known at a level Current Opinion in Plant Biology of detail that will rival what is known about . Exon Intron structure of the APR genes. The genomic sequence from the translational initiation codon to the termination codon is shown as a diagram. The solid bars reflect the coding exons and the Note added in proof open bars reflect introns. The dotted lines indicate the analogous The paper referred to in the text as (Bick and Leustek, introns in each gene. The solid circle shows the position of the 12 basepair direct repeat sequences. The open circle shows the unpublished data) has now been accepted for publication •• approximate border between the sequence encoding the reductase [37 ]. and thioredoxin/glutaredoxin domains. References and recommended reading same position in APR2 and APR3. It is an intriguing Papers of particular interest, published within the annual period of review, have been highlighted as: possibility that this repeat is the remnant of an intron that divided the last exon of these genes, isolating the • of special interest •• of outstanding interest thioredoxin/glutaredoxin-like domain in a separate exon. 1. Brunold C, Rennenberg H: Regulation of sulfur metabolism in • plants: first molecular approaches. Prog Bot 1997, 58:164-186. Contribution of APR gene expression to the An excellent recent review on the topic of plant sulfur metabolism. regulation of sulfate assimilation 2. Hell R: Molecular physiology of plant sulfur metabolism. Planta Although the sulfate assimilation pathway in plants • 1997, 202:138-148. • appears to be regulated, the published evidence is not very See annotation [1 ]. clear on which steps are most important. This is in contrast 3. Schwenn J: Assimilatory reduction of inorganic sulphate. In • Sulphur Metabolism in Higher Plants: Molecular, Ecophysiological to the situation in E. coli where the genes encoding all and Nutritional Aspects. Edited by Cram WJ, De Kok LJ, Stulen I, the pathway enzymes are co-ordinately regulated by gene Brunold C, Rennenberg H. Leiden: Backhuys Publishers; 1997:39- 58. expression in response to the availability of cysteine [13]. See annotation [1•]. Thus, we must consider whether regulation in plants 4. Varin L, Chamberland H, Lafontaine JG, Richard M: The enzyme might be highly co-ordinated at the level of tissues and •• involved in sulfation of the turgorin, gallic acid 4-O-(β-D- cells rather than at the whole plant level. Recently, the first glucopyranosyl-6′-sulfate) is pulvini-localized in Mimosa pudica. Plant J 1997, 12:831-838. attempt at defining the specific cells and tissues in which An important contribution to the field of sulfation in plants, a poorly under- sulfate assimilation genes are expressed was published stood area of sulfate metabolism. [30••]. In the future, more work of this kind is needed. 5. Varin L, Marsolais F, Richard M, Rouleau M: Biochemistry and •• molecular biology of plant sulfotransferases. FASEB J 1997, 11:517-525. APSSTase has long been the focus of investigation See annotation [4••]. because it is believed to be a key point for regulation 6. Br¨uhl A, Haverkamp T, Gisselmann G, Schwenn JD: A cDNA clone of the sulfate assimilation pathway. Most importantly, the from Arabidopsis thaliana encoding plastidic ferredoxin:sulfite reductase. Biochem Biophys Acta 1996, 1295:119-124. published literature indicates that changes in enzyme 7. Bogdanova N, Hell R: Cysteine synthesis in plants: protein- level are the primary means by which APSSTase activity •• protein interactions of acetyltransferase from is regulated [31]. Recent investigations on the affect of Arabidopsis thaliana. Plant J 1997, 11:251-262. Not discussed in our review is the topic of complex formation between O- sulfate starvation on A. thaliana showed that the steady acetylserine (thiol) lyase and serine acetyltransferase, which together form state level of mRNA for sulfate permease and the APR cysteine synthetase. The function of this complex is not understood. This is enzymes increases markedly in and to a lesser extent the first attempt to define the complex at the molecular level. in [32•]. The mRNA level for all of the other sulfate 8. Schmidt A, Jager¨ K: Open questions about sulfur metabolism in plants. Annu Rev Plant Physiol and Plant Molec Biol 1992, assimilation genes was not increased or, in some cases, 43:325-349. it decreased. This result is consistent with the idea that 9. Schwenn JD: Photosynthetic sulfate reduction. Z Naturforsch APSSTase and APS reductase are the same enzyme and 1994, 49:531-539. 244 Physiology and metabolism

10. Li J, Schiff JA: Purification and properties of adenosine 5′- 27. Speich N, Dahl C, Heisig P, Klein A, Lottspeich F, Stetter KO, phosphosulfate sulfotransferase from Euglena. Biochem J Trupper ¨ HG: Adenylylsulphate reductase from the sulphate- 1991, 274:355-360. reducing archaeon Archaeoglobus fulgidus: cloning and characterization of the genes and comparison of the enzyme 11. Schiffmann S, Schwenn JD: APS-sulfotransferase activity is with other iron-sulphur flavoproteins. Microbiol 1994, identical to higher plant APS-kinase (EC 2.7.25). FEBS Lett 140:1273-1284. 1994, 355:229-232. Three genomic clones from Arabidopsis Reaction 28. Chen Y, Leustek T: 12. Berendt U, Haverkamp T, Prior A, Schwenn JD: • thaliana encoding 5′-adenylylsulfate reductase (Accession mechanism of thioredoxin: 3′-phospho-adenylylsulfate Nos. AF016282, AF016283 and AF016284). Plant Physiol 1997, reductase investigated by site-directed mutagenesis. Eur J 116:447. Biochem 1995, 233:347-356. 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