Bioinformatic and enzymatic characterization of the MAPEG superfamily Anders Bresell 1, *, Rolf Weinander 2, *, Gerd Lundqvist 3 , Haider Raza 3 , Miyuki Shimoji 3 , Tie-Hua Sun 3 , Lennart Balk 5 , Ronney Wiklund 6 , Jan Eriksson 6 , Christer Jansson 6 , Bengt Persson 1,4 , Per-Johan Jakobsson 2 and Ralf Morgenstern 3 1 IFM Bioinformatics, Linko ¨ ping University, Sweden 2 Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden 3 Institute of Environmental Medicine Karolinska Institutet, Stockholm, Sweden 4 Centre for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden 5 Stockholm Marine Research Centre, University of Stockholm, Sweden 6 Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala, Sweden Keywords MAPEG; microsomal glutathione transferase; prostaglandin; leukotriene Correspondence R. Morgenstern, Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden Fax: +46 8 343849 Tel: +46 8 5248 7574 E-mail: ralf.morgenstern@imm.ki.se *Both authors contributed equally to this work (Received 15 November 2004, revised 27 January 2005, accepted 3 February 2005) doi:10.1111/j.1742-4658.2005.04596.x The membrane associated proteins in eicosanoid and glutathione metabo- lism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belong- ing to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calcula- tions of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal gluta- thione transferases (MGST) 1, 2 and 3, leukotriene C 4 synthase (LTC 4 ), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2 ⁄ FLAP ⁄ LTC 4 synthase families. The insect members are most similar to MGST1 ⁄ prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, show- ing an early origin for MAPEG family differentiation. Thus, the evolution- ary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the sub- families. We have further investigated and functionally characterized repre- sentative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity ana- lysis demonstrated that all proteins catalyzed the conjugation of 1-chloro- 2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 lmolÆmin )1 Æmg )1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 lmolÆmin )1 Æmg )1 . Yeast microsomes expressing the Arabidopsis enzyme Abbreviations BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; DEAE, diethylaminoethyl; FLAP, 5-lipoxygenase activating protein; LT, leukotriene; MGST, microsomal glutathione transferase; PG, prostaglandin; PGES, prostaglandin E synthase; GST, glutathione S-transferase; GPx, glutathione peroxidase; CuOOH, cumene hydroperoxide. 1688 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS Microsomal glutathione transferases (MGSTs) repre- sent a recently recognized superfamily of enzymes involved in detoxification, but also in specific biosyn- thetic pathways of arachidonic acid metabolism. The superfamily was termed the membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) and consists of proteins from mammals, plants, fungi and bacteria [1]. The six members in humans include 5-lipoxygenase activating protein (FLAP) and leukotriene (LT) C 4 synthase, which are both involved in leukotriene biosynthesis [2,3]; MGST1, MGST2 and MGST3, which all are gluta- thione transferases as well as glutathione dependent peroxidases [4–7]; and finally, prostaglandin (PG) E synthase (PGES), earlier referred to as MGST1-L1 [8]. PGES catalyzes the formation of PGE 2 from PGH 2 , which in turn is generated from arachidonic acid by the prostaglandin endoperoxide synthase systems. PGES has also been referred to as p53 induced gene 12 (PIG12) because the gene expression was found to increase extensively following p53 expression [9]. The relationships and other functional aspects of the MAPEG enzymes have been reviewed [10]. Two groups of bacteria, purple bacteria and cyano- bacteria, have been found to produce and maintain significant levels of glutathione [11] and, interestingly, also contain MAPEG members [1]. Glutathione was observed in various species within the two groups, among those in Escherichia coli, one of the most well characterized species of purple bacteria [11]. The func- tion of glutathione metabolism in bacteria may be pro- tection against xenobiotics and ⁄ or oxidative stress but also as part of specific biosynthetic pathways [12]. Cyanobacteria produce oxygen by photosynthesis and purple bacteria can use oxygen as a terminal electron acceptor. Glutathione production in bacteria is thus closely associated with those bacteria that generate or utilize oxygen in specific biochemical pathways indica- ting that glutathione metabolism originated in bacteria at the time when an oxygen-containing atmosphere developed on earth [11,12]. A low level of glutathione S-transferase (GST) activ- ity has been demonstrated in E. coli but not in cyano- bacteria [11]. Cytosolic GSTs have been identified in various strains of bacteria [12] and in a few studies, including those on Proteus mirabilis and E. coli, cyto- solic GSTs have been purified and further character- ized [13–15]. The three-dimensional structure of the P. mirabilis cytosolic GST has also been determined [16]. In Synechocystis sp. a gene homologous to cyto- solic GST exists but has not been characterized further [17]. In general, the enzymes involved in glutathione metabolism in prokaryotes have not been so exten- sively studied and therefore less is known about their properties as compared to the corresponding proteins in eukaryotes. Microsomal GST activity has not been demonstrated in any prokaryotic organism. Expressed sequence tag (EST) clones with open reading frames (ORFs) similar to MAPEG proteins have been found in E. coli, Synechocystis sp. and Vibrio cholerae [1]. The Synechocystis sp. ORF dis- played sequence similarity to the MAPEG subfamily consisting of FLAP, LTC 4 synthase and MGST2, and also to the MGST3 subfamily but it could not be sig- nificantly grouped to any of those two subfamilies, whereas the E. coli and V. cholerae sequences form a separate group [1]. Nothing is known, however, about the enzymatic properties of any prokaryotic MAPEG protein. As the number of sequenced bacterial genomes has increased considerably during recent years, we de- signed this study to search further for MAPEG pro- teins and functionally characterize representative gene products. Database searches revealed various new gene products, in some cases coexisting, with homologies to the two MAPEG subfamilies (described above and in [1]). We investigated representative gene products from the E. coli and Synechocystis sp. bacteria further, to gain insight into the function of these proteins and the evolution of the MAPEG superfamily. Cloning and overexpression demonstrated that both are membrane- bound glutathione transferases. showed an activity of 0.02 lmolÆmin )1 Æmg )1 , whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 lmolÆmin )1 Æmg )1 . The purified pike enzyme is the most active MGST described so far with a spe- cific activity of 285 lmolÆmin )1 Æmg )1 . Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 lmolÆmin )1 Æmg )1 , respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES sub- families. A. Bresell et al. Characterization of MAPEG members FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1689 To understand the evolutionary relationships better on a more global scale we also cloned and expressed (or purified) MAPEG representatives from plant, insect and fish. Together with earlier data on the frog enzyme [18] these data define glutathione transferase activity as a central property of MAPEG members from a wide range of organisms and suggest ancestral MAPEG members. Results MAPEG members from complete genomes Over 130 MAPEG members were retrieved from sequence databases and completed genomes, of which less than half (56) were previously known members according to the PF01124 entry in Pfam release 11 [19]. Multiple sequence alignments and hydrophobicity plots were calculated (for a full alignment see supplementary Fig. 1). Even though several members are distantly related, all exhibit the typical MAPEG properties of 150 residue subunits with four hydrophobic regions, compatible with four transmembrane regions [20,21]. Using information from completed genomes, we have traced the evolutionary relationships of the MAPEG members. The general relationships are depicted in Fig. 1. MGST1, PGES and insect forms have a common branch, compatible with their overlap- ping substrate-specificities [22]. Likewise, MGST2, FLAP and LTC 4 synthase also show somewhat closer relationships, indicating properties in common. MGST3 forms a separate branch. The bacterial E. coli and Synechocystis variants are found on separate bran- ches. A detailed dendrogram is shown in Fig. 2. The bacterial forms show distant relationships and their exact grouping is not significant at all sites, as indicated from their low bootstrap values (no asterisks in Fig. 2). Furthermore, the bacterial forms are present at three sites in the dendrogram. However, the group- ing of the families MGST1, MGST2, MGST3, PGES, FLAP and LTC 4 synthase is significant. In a dendro- gram without the bacterial forms, the grouping of these families becomes even more evident (not shown). Among the MAPEG sequences from fish, we find members from all six branches (MGST1, MGST2, MGST3, PGES, FLAP and LTC 4 synthase), suggest- ing that the origin of these forms dates back to before the occurrence of vertebrates, i.e. more than 500 mya. This dates the differentiation of the MAPEG forms back to the late Cambrian multiplicity of eukaryotic species. Notably, in the screenings we have not found any members from the archaea kingdom, indicating that the enzymatic activities of the MAPEG family are not present in these species or that these activities are catalysed by other enzymes. The absence of MAPEG members in archaea is certainly consistent with the lack of GSH in these organisms. Cloning, expression and characterization of selected MAPEG members MGST homologues from Synechocystis and E. coli After identifying MAPEG members in several bacterial strains, the E. coli and Synechocystis sp. proteins were Fig. 1. Schematic evolutionary tree of the MAPEG superfamily. The evolutionary tree shows the relationships between the six MAPEG families and three further groups (Insect, E.coliMGST cluster and SynMGST cluster). A major subgrouping is visible with MGST1, PGES and Insect in the upper part of the tree and the remaining families ⁄ groups in the lower part. In the lower part, MGST2, FLAP and LTC4 synthase have a close relationship, as judged by the short branches between these enzymes. Fig. 2. Detailed dendrogram of the MAPEG superfamily. The tree shows all presently known MAPEG forms, excluding species variants which differ at only a single position. In the tree, the six families are clearly distinguished. The prokaryotic forms are found at three sites – the E. coli cluster, the Synechocystis cluster, and the group of remaining forms, denoted Bacteria. Two further groups are marked, denoted Insects and Waterliving. The branch lengths are proportional to the number of residue differences, with the scale bar indicating a 5% amino acid difference. The fish forms, having representatives for all six MAPEG families, are marked with a fish symbol. Accession numbers refer to the databases Uniprot, NCBI or ENSEMBL. Characterization of MAPEG members A. Bresell et al. 1690 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS A. Bresell et al. Characterization of MAPEG members FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1691 selected for functional characterization of bacterial MGST homologues. These homologues represent two different groups of prokaryotic MAPEG members found. The E. coli ORF, which we refer to as E.coliMGST, encodes a 141 amino acid residue polypeptide with a cal- culated molecular mass of 16.2 kDa. The Synechocystis sp. ORF (from strain PCC6803 [23]) encodes a 137 resi- due polypeptide with a predicted molecular mass of 15.4 kDa, which we refer to as SynMGST. The ORFs encoding E.coliMGST and SynMGST were amplified by PCR, the products cloned into an expression vector and the DNA sequences were verified against the EMBL database entries. Following hetero- logous expression in E. coli, the membrane fractions were assayed for enzyme activities. The membrane fraction from cells overexpressing E.coliMGST cata- lyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione with a specific activity of 0.11 lmolÆmin )1 Æmg )1 . When a shorter con- struct beginning from the alternative translation start site of the E.coliMGST was expressed no activity was detected. Incubation with N-ethylmaleimide (which activates mammalian MGST1) did not affect the activity of E.coliMGST. Membranes from cells over- expressing the SynMGST also displayed glutathione transferase activity. The glutathione conjugating activ- ity towards CDNB was 1.7 lmolÆmin )1 Æmg )1 for the SynMGST membrane fraction. Neither LTC 4 synthase activity, nor any glutathione-dependent peroxidase activity (towards cumene hydroperoxide or 5-hydrope- roxy-eicosatetraenoic acid) could be observed in any of the fractions. No activity could be detected with these enzymes towards 1,2-epoxy-3-para-nitrophenoxypro- pane or trans-phenylbut-3-en-2-one as substrates (sum- marised in Table 1). Partial purification of SynMGST To characterize bacterial MGSTs further we concen- trated on SynMGST. RT-PCR was used to confirm that SynMGST is indeed expressed in the cyanobac- teria (Fig. 3). Having established gene expression of SynMGST in the cyanobacteria and a functional overexpression of recombinant protein in E. coli we made an attempt to purify the protein for further characterization. Bacterial membranes isolated from cells overexpressing recom- binant SynMGST were solubilized in Triton X-100. The recombinant SynMGST was also enzymatically active upon detergent solubilization and the CDNB conjugating activity was used to monitor subsequent purification steps. The SynMGST is basic (the cal- culated isoelectric point being 9.9) and could therefore be expected to yield a purified product using meth- ods developed for MGST1 [24]. However, although the enzyme behaved in a predictable manner upon hydroxyapatite batch chromatography, in cation exchange chromatography the enzyme was recovered in the flow-through fractions. Diethylaminoethyl (DEAE) columns, likewise, did not retain the enzyme. Because cation and anion exchange chromatography, in concert, did retain most of the contaminating proteins, a parti- ally purified protein was nevertheless recovered. In fact, SDS ⁄ PAGE (Fig. 4) shows that the protein is nearly homogeneous. Furthermore, N-terminal amino acid Table 1. Comparison of glutathione transferase and peroxidase activity of MAPEG members expressed ⁄ purified from prokaryotes, plant, nonmammalian and mammalian species. ND, not detectable. Species CDNB activity (lmolÆmin )1 Æmg )1 ) CuOOH GPx activity (lmolÆmin )1 Æmg )1 ) Activity of purified enzyme Human MGST1 [71,72] 1.9 0.04 Rat MGST1 [43] 2 0.08 Xenopus laevis, frog [18] 210 2.1 Esox lucius, pike 290 2.2 Synechocystis sp. 11 ND (partially purified) Activity in membrane fraction after heterologous expression Drosophila melanogaster 3.6 0.4 Arabidopsis thaliana 0.02 n.d. Synechocystis sp. 1.7 n.d. Escherichia coli 0.11 n.d. 800 12 34 400 200 100 Fig. 3. RT-PCR. To demonstrate that the SynMGST gene was expressed in Synechocystis 6803, total RNA was isolated and amplified by PCR with SynMGST-specific primers, in the presence (lane 4) or absence (lane 3) of reverse transcriptase. PCR amplifica- tion from isolated total DNA, using the same primers (lane 2) served as a positive control. Sizes in bp, deduced from a 100 bp ladder (lane 1) are indicated. Characterization of MAPEG members A. Bresell et al. 1692 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS sequence analysis of the predominant band displaying the correct molecular mass, purified from the gel, yielded the expected sequence. The partially purified protein constitutes a major part of the preparation and therefore the specific activities measured will be close to those of the pure enzyme. The enzyme is more active than its mammalian counterparts and expressed extremely well. Assuming that the protein was at least 50% pure, the purification factor (12-fold) indicates that expressed SynMGST constituted about 8% of the E. coli membrane asso- ciated proteins. The specific activity of the partially purified enzyme with 1-chloro-2,4-dinitrobenzene was 11 ± 0.4 lmolÆmin )1 Æmg )1 (mean ± SD, n ¼ 3). The activity was not affected by incubation with the sulf- hydryl reagent N-ethylmaleimide in contrast to mam- malian MGST1, which is activated several-fold by this reagent. MGST3 from Arabidopsis When plant MGST3 was cloned and overexpressed in a yeast expression system, the yeast microsomes displayed a low glutathione transferase activity with CDNB (0.02 lmolÆmin )1 Æmg )1 ) that was not activa- ted ⁄ inhibited by N -ethylmaleimide. Glutathione peroxi- dase activity was not altered compared to that in microsomes from yeast expressing the pYeDP60 vector only (the negative control). MGST1/PGES-like enzyme from Drosophila The MGST from Drosophila was cloned and over- expressed in E. coli where the isolated membrane fraction displayed a high glutathione transferase acti- vity (3.6 lmolÆmin )1 Æmg )1 ) and glutathione peroxidase activity (0.4 lmolÆmin )1 Æmg )1 ). Addition of 1% (v ⁄ v) Triton X-100 to the membrane fraction resulted in a slight increase in activity, whereas N-ethylmaleimide had no effect on enzyme activity. The enzyme did not display PGES activity. MGST1/PGES-like enzyme from pike MGST was successfully purified to apparent homogen- eity (Fig. 4) from pike liver using protocols developed for the rat enzyme. The N-terminal sequence of the purified pike enzyme was determined using Edman de- gradation. Sequence comparisons reveal that the pike form purified is closely related to the MGST1 ⁄ PGES branch (Fig. 5). Of the N-terminal 47 residues, 22–28 residues are identical to fish MGST1 sequences, while only 2–12 residues are identical to the fish sequences of other MAPEG families. The enzymatic properties of the pike MGST1-like enzyme were extensively characterised (Table 1) dem- onstrating that the protein has the highest glutathione transferase activity of any MAPEG member detected so far. As the enzyme displays similar substrate speci- ficity to MGST1, including glutathione peroxidase activity, the assignment to the MGST1 ⁄ PGES sub- family appears well founded. Sequence patterns of the MAPEG members For the MGST1–3, FLAP, LTC 4 synthase, PGES and Insect family clusters we generated sequence patterns, shown in Table 2. These patterns are all 100% unambiguous when scanned against Swiss-Prot and TrEMBL, i.e. no nonmembers are ranked higher than Syn MGST MGST MGST1 Rat Pike 1mg/lane 1mg/lane MGST1 Rat 75 ng 25 ng kDa Mr markers kDa Mr markers 45 31 21.5 14.4 10 20 15 150 ng Fig. 4. SDS ⁄ PAGE analysis of purified SynMGST and pike MGST. The protein was fractionated on SDS ⁄ PAGE (15%) and visualized by silver staining. Major proteins were detected that comigrated with purified RatMGST1 (17 kDa). A. Bresell et al. Characterization of MAPEG members FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1693 the lowest ranked true member. These patterns are more specific then the existing PROSITE pattern PS01297 (FLAP ⁄ GST2 ⁄ LTC4S: G-x(3)-F-E-R-V-[FY]- x-A-[NQ]-x-N-C) [25]. The patterns are selected based on conserved regions in the sequence. Notably, the PGES pattern is located at the beginning of loop one and for FLAP it is located in the third hydrophobic seg- ment. All of the remaining patterns are located at the end of first loop (Fig. 6). Both the first and third loop are located on the cytosolic side of the membrane and are regions earlier postulated to host the active site [21,26]. Furthermore, the patterns of the two very sim- ilar families of PGES (earlier denoted MGST1-like) and MGST1 do not overlap, even though they both are located in the first loop. For the classical FERV pattern, which is a part of PS01297, we note that it is still included in the two new and more specific patterns of MGST2 and LTC 4 synthase. The last member of PS01297 is FLAP for which the novel pattern is located in the third loop. The reason for the similarity and location of these pat- terns could be a result of short evolutionary time rather than gain of new features as FLAP, MGST2 and LTC 4 synthase have been detected only in higher eukaryotes to date. However, all patterns in Table 2 will be useful in genome characterizations and func- tional annotations. Discussion The MAPEG family We have characterized the MAPEG family and found the eukaryotic forms to consist of six families, while the prokaryotic forms are clustered at two sites or more, depending upon whether the E. coli cluster (top) and the bacterial cluster (bottom) are separated or not (Fig. 2). The SynMGST branches with the cluster of Fig. 5. Alignment of pike MGST1 with homologous forms. The N-terminal fragment of pike MGST1 is multiply aligned with other MAPEG fish members. Positions identical between the pike form and any other fish form are shown in bold. It can be seen that most of the bold amino acid residues are found within the MGST1 family, supporting evidence for the pike form to belong here. A dendrogram is shown to the left of the alignment, calculated from the aligned sequences. Table 2. Sequence patterns for the different MAPEG families. Family Pattern Position FLAP P-A-A-F-A-G-x(0,1)-L-x(0,1)-Y-L-x(2)-R-Q-K-Y-F-V-G-Y 123 LTC 4 synthase G-P-P-E-F-[DE]-R-[IV]-[FY]-R-A-Q-[AV]-N-[CS]-[ST]-E-Y-F-P 66 MGST1 E-R-V-R-R-[ACG]-H-x-N-D-[IL]-E-N-[IV]-[IV]-P-F-[FLV]-[AGV]-I 92 MGST2 V-[ST]-G-[APS]-[LP]-[DE]-F-[DE]-R-x-F-R-A-x(0,1)-Q-x(0,1)-N-[CNS]-[ALV]-E 63 MGST3 F-N-C-[AIV]-Q-R-[AGS]-H-[AQ]-[NQ]-x(2)-E-x(2,3)-P 90 PGES M-Y-[AIV]-[IV]-A-[IV]-I-T-G-Q-[IMV]-R-L-R-[KR]-K-A-x-A-N 47 Insect D-P-x-V-E-R-V-R-R-A-H-x-N-D-x-E-N-I-L-P 87 Characterization of MAPEG members A. Bresell et al. 1694 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS FLAP, MGST2 and LTC 4 synthase, while E.coli- MGST branches earlier, before the divergence of MGST3 from the previous cluster. However, it should be kept in mind that the early branches have low boot- strap values and that the order might change when more data become available. Interestingly, several bac- terial species contain multiple MAPEG forms. For example, the Caulobacter crescentus has three different forms – one in the SynMGST cluster, one in the Ecoli- MGST cluster and one in the large bacterial cluster (Fig. 2). We checked whether any of the MAPEG members were encoded by plasmids, but we did not find any MAPEG members among known plasmid sequences. Mutiplicity of MAPEG members is also seen in insects. Both Drosophila and Anopheles show multiple forms, but these forms are more closely related than the multiple forms of bacterial species. As judged from sequence comparisons, the insect multiple forms have appeared independently in each species, probably reflecting adoption to the environment. Interestingly, Drosophila also has multiple gene families of cytosolic GSTs [27]. Extensive searches in archaea only revealed possible homologues related to transport proteins. If these rela- tionships are real they might give a link to ancestors with different functions, which were later recruited as detoxification enzymes. Upon examination of the eukaryotic MAPEG forms, we found that the subdivision into six different families is present already in fish, dating this diver- gence to 500 mya. These findings agree in general with the known well developed capacity of fish xenobiotic metabolism [28] and raises the possibility of arachi- donic acid based signalling. Zebrafish express both cyclooxygenase (cox)-1 and -2 and the primary prostaglandin end product is PGE 2 [29]. Furthermore, the bleeding time as a measurement of platelet activa- tion was sensitive to inhibition of cox-1 but not of cox-2, i.e. similar to the situation in humans. Incuba- tion of whole blood from rainbow trout with calcium ionophore resulted in the biosynthesis of leuko- triene B 4 suggesting an intact leukotriene pathway including phospholipase, 5-lipoxygenase and LTA 4 hydrolase in this species [30]. Thus, the fish kingdom seems to contain a similar biosynthetic capacity to humans to oxidize arachidonic acid. In plants, leuko- triene B 4 has been demonstrated in nettles [31] prob- ably as part of its defence mechanism. In various species of corals, large amounts of prostaglandin-rela- ted compounds are found [32]. Here the prostaglandin- like compounds may constitute structural elements of the organism or be part of their chemical defence. Recently, two coral (Gersemia fruticosa) cyclooxygen- ases were cloned and functionally characterized, and found to catalyze the formation of PGF 2a , PGE 2 and PGD 2 (presumably through nonenzymatic conversion of PGH 2 ) as well as unspecified hydroxyeicosatetra- enoic acids [33]. It is also suggested that an ancestral gene coding for cyclooxygenase was duplicated before A B Fig. 6. Hydrophobicity plot. (A) The plot shows the mean value of hydrophobicity (solid lines) and standard deviation (dashed lines). Values are calculated according to Kyte and Doolittle [53] using an 11-residue window. The positional numbers follow a multiple sequence alignment of all MAPEG members. (B) The plot shows the number of sequences present at each position. The four hydrophobic segments, corresponding to the transmembrane regions are visible as peaks in (A). A. Bresell et al. Characterization of MAPEG members FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1695 the divergence of the modern cyclooxygenase-1 and -2. It would be interesting to know at what time during development the MAPEG proteins (specifically PGE synthase and FLAP ⁄ LTC4 synthase) were associated with the cyclooxygenase and lipoxygenase protein families, respectively. At the introduction of these MAPEG proteins a more specialized level of product control must have occurred, allowing for the specific metabolism of the products derived from cyclooxygen- ases and lipoxygenases into the end products known today. Structural implications Now that over 100 different MAPEG forms are avail- able, a limited number of conserved residues have appeared. Two of these, Glu81 and Arg114 (human MGST1 positional numbering), are found in the puta- tive transmembrane segments 2 and 3, respectively. According to electron crystallographic structure deter- mination of MGST1 [34] and LTC4 synthase [35] and hydrophobicity properties, the MAPEG forms all appear to contain four transmembrane regions. MGST1, LTC4 synthase and PGES [22] are all trimeric proteins. At the tight border between transmembrane region 2 and 3, some of the sequences have a Gly-Pro sequence, typical of a sharp bend. Interestingly, the almost strictly conserved charged residues mentioned above are both spaced by exactly 15 residues from the Gly-Pro bend, strengthening a role for structural charge interactions. In addition, Asn78 is conserved in almost all MAPEG members. This residue faces the cytosol, positioned just before the second transmem- brane segment, and is probably involved at the active site. In fact, mutation of these residues in MGST1 seriously affects activity (unpublished observations). Mutation of the residue corresponding to Arg114 (Arg110) in human mPGES-1 also abolishes activity [36]. Similarly Arg130, facing the cytosol and adjacent to the fourth transmembrane segment, is conserved in nearly all members. The sequence patterns diagnostic for the PGES and FLAP families are both found in regions facing the cytosol, thus implying that they represent family specific regions of the active site and ⁄ or substrate-binding areas. Observations on the proteins E.coliMGST and SynMGST represent the first charac- terized prokaryotic members of the MAPEG super- family. It was therefore of strong interest to determine their catalytic properties. Both enzymes efficiently cata- lyze a glutathione transferase reaction and conse- quently may be involved in detoxification. In contrast to human MGSTs 1, 2 and 3, no glutathione peroxi- dase activity could be detected. Our results thus demonstrate that both of these highly divergent pro- karyotic MAPEG members indeed are microsomal glutathione transferases. SynMGST, MGST2, and LTC 4 synthase to some extent, align with a postulated lipid binding site of FLAP (amino acids 48–61) [37–39]. In addition, Syn- MGST contains conserved arginine and tyrosine resi- dues implicated in LTC 4 synthase activity [40]. However no such activity could be detected, logically coinciding with the fact that 5-lipoxygenase (forming the substrate) as well as other lipoxygenases are found later in evolution [41]. However, recently a 15-lipoxy- genase was characterized as a secretable enzyme in Pseudomonas aeruginosa [42] and is, to the best of our knowledge, the first example of a lipoxygenase in bacteria. The cyanobacteria, Synechocystis spp., represent an interesting model system for further studies of the bio- logical functions of SynMGST. Knock out experi- ments, as well as studies of the effects caused by environmental factors such as light and oxygen on SynMGST gene expression, will provide important information about the biological function. Moreover, if the MGSTs represent common bacterial components involved in glutathione metabolism mediating cell sur- vival, they may constitute possible targets for the development of novel antibiotics. N-ethylmaleimide, activity and activation Mammalian MGST1 is activated by sulfhydryl rea- gents and its relatively modest activity towards CDNB is increased by 20-fold (from 3 lmolÆmin )1 Æmg )1 to 60 lmolÆmin )1 Æmg )1 ) [43]. An MGST has been purified from Xenopus laevis that was extremely active (200 lmol min )1 Æmg )1 ) but on the other hand very sen- sitive to sulfhydryl reagents [44]. The pike enzyme is also inactivated by N-ethylmaleimide (not shown). Synechocystis, Arabidopsis and Drosophila MGSTs appear to represent a third category, namely enzymes that are insensitive to sulfhydryl reagents. In the case of Synechocystis and Drosophila enzymes, this is accounted for by the fact that no cysteine residues are present and probably explains why SynMGST is an exceptionally stable protein (in our experience). The catalytically active form of E.coliMGST contains three cysteine residues but was not activated by N-ethylmaleimide. Instead a slight inhibition of the activity towards CDNB was observed. Apparently, none of the cysteines is situated at an accessible posi- Characterization of MAPEG members A. Bresell et al. 1696 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS tion that is critical for enzyme activity of the E.coliMGST. In conclusion, sulfhydryl reagent activa- tion ⁄ inactivation cannot be used as a criterion to iden- tify MAPEG MGST1 members as the activation has been detected so far only with mammalian MGST1. Also, the closest relative of MGST1, PGES, is inacti- vated by N-ethylmaleimide [22] as well as LTC 4 syn- thase [45]. It is evident that cysteine is not involved in the catalytic mechanism of several MAPEG members, but could well be relevant for PGES and LTC 4 syn- thase, which harbour cysteines at unique positions. Conclusion We have identified several new MAPEG proteins by sequence homologies with proteins in various databases. The mammalian members can be traced back 500 mya as all six families can be found in fish, consistent with a role in eicosanoid signalling. The gene products from two representative bacterial strains, E. coli and Synechocystis sp. were cloned and overexpressed in E. coli. In addition, plant, insect and fish MAPEG mem- bers were characterized. As a common denominator, most MAPEG members catalyze glutathione conjuga- ting activity towards CDNB or a specific substrate such as LTC 4 , some with remarkable efficiency. The enzymes represent early MAPEG members in their respective phylogenetic classes and thus create a defined basis for understanding this superfamily. Experimental procedures Materials Oligonucleotides were synthesized by KEBO, (Stockholm, Sweden). Pfu DNA polymerase was purchased from Strata- gene (La Jolla, CA, USA). pGEM T-vector was from Promega (Madison, WI, USA). Gel extraction and plasmid isolation kits were from Qiagen (Hilden, Germany). DNA sequencing kit (ABI PRISM Dye Terminator Cycle Sequen- cing Ready Reaction Kit) was obtained from Perkin-Elmer (Boston, MA, USA). Hydroxyapatite (Bio-Gel HTP) was from Bio-Rad (Hercules, CA, USA). Sequence comparisons In the search for new members of the MAPEG superfamily a set of representative members were selected as seeds. The seeds were the human member proteins of MGST1-3 (Uni- prot-Swissprot identifiers P10620, Q99735 and O14880); FLAP (P20292); LTC 4 synthase (Q16873) and PGES (O14684). Two bacterial members, SynMGST (P73795) and E.coliMGST (P64515), were additionally selected to com- plement the six human forms. The eight seeds were used as query sequences in the search for homologues using fasta [46] against Swissprot release 41.24 [47], TrEMBL release 24.13 [47] and 138 completely sequenced genomes. Further screenings were performed against the NCBI non-redund- ant protein database using psi-blast [48]. Finally, to fetch unverified translations of MAPEG members the NCBI EST database (excluding human and mouse) [49] was searched using tblastn [48]. The resulting nucleotide sequences from the EST search were translated using getorf from the emboss package [50]. The open reading frames were filtered by a minimum size of 100 amino acid residues and flanked by start and stop codons. These homology searches resulted in nearly 1000 redundant amino acid sequences which were followed by an extensive work of manual filtering to obtain a non-redundant set of sequences by removing duplicates and non-EST supported alternative splicings. Multiple sequence alignments and dendrograms To study the relationships between the new members of the superfamily we calculated multiple alignments using clu- stalw [51] on the resulting sequences from the homology searches. Dendrograms were obtained using neighbor-join- ing method in the clustalw package and protpars from the phylip package [52]. An unrooted tree was generated based on the complete set of sequences of all superfamily members. To also visualise the more general relationships of the families included in MAPEG an unrooted consensus tree was produced. The consensus sequences of the families of MGST1–3, FLAP, LTC 4 synthase, PGES, SynMGST cluster, E.coliMGST cluster and Insect cluster were gener- ated by the cons program from the emboss package. A hydrophobicity plot was generated to verify the structural similarities of the proteins. It was based on the multiple sequence alignment of the complete superfamily and calcu- lated according to Kyte and Doolittle [53] using a window of 11 residues. Pattern detection To characterize the MAPEG families further we extracted patterns compatible to the PROSITE database [25,54]. These patterns are helpful in annotation of new sequences and model the unique motifs of a family. The patterns were generated by the program pratt version 2.1 [55,56]. pratt was run on sequences from each of the MGST1-3, FLAP, LTC 4 synthase, PGES and Insect families by setting the maximal pattern length parameter to 20. The best ranked patterns of each family, shown in Table 2, were selected and tested for unambiguousness by performing a scan against Swiss-Prot and TrEMBL with the program fuzz- pro from the emboss package. The degree of unambiguous- ness was defined as the fraction of member ranked higher than the first occurring non-member. A. Bresell et al. Characterization of MAPEG members FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS 1697 [...]... negative controls, DNA and RNA were used as templates, respectively, without the addition of reverse transcriptase Preparations of Synechocystis 6803 DNA and RNA were performed as described previously [57] Isolation and cloning of the SynMGST and E.coliMGST The coding sequence for the SynMGST, corresponding to the complementary strand of the nucleotide sequence from 89 254 to 89 667 in the Synechocystis... BamHI and EcoRI and then the reformatted BamHI-EcoRI fragment was subcloned into BamHI and EcoRI sites of the pYeDP60 vector The nucleotide sequence of the cDNA was determined with a PerkinElmer model 373 automated sequencer using forward primer 1 and reverse primer 1 No mutation was detected The transformed yeast cells were cultured and the microsomes were prepared by differential centrifugation The. .. was retained on either ion exchanger, the protein content of the flow-through from the DEAE-Sephadex was examined by SDS ⁄ PAGE A predominant protein band, comigrating with rat MGST1, was observed This band was also observed in fractions from the other purification steps The band was cut out from the gel followed by elution of the protein and the N-terminal amino acid sequence was determined using an... ampicillin (75 lgÆmL)1) and chloramphenicol (10 lgÆmL)1) in a 5 L flask placed in a thermostated water bath The culture was oxygenated by air bubbling and grown until the D600 was 0.4–1.2 Expression was then induced by the addition of 0.4 mm isopropyl thio-b-d-galactoside, the temperature was switched to 30 °C and the culture allowed to grow for another 4 h Thereafter, cells were pelleted and resuspended in... upstream of the initiation codon (start of uppercase) were designed as follows: forward primer 2, 5¢-cgggatccATGG CGGCGATTACAGAATTTC-3¢ To obtain the cDNA with suitable restriction sites, forward primer 2 and T7 primer were used for PCR with Pfu DNA polymerase as the pGEMT Easy vector has an EcoRI site downstream of the stop codon of MGST3 The PCR product was digested with 1699 Characterization of MAPEG. .. Finally, the AMV reverse transcriptase was heat inactivated at 95 °C and the extension product was PCR amplified by the addition of sense primer and 2.5 units of Taq polymerase (Pharmacia Biotech) The temperature cycles were 95 °C, 1 min, 55 °C, 1 min and 72 °C 1 min, repeated 25 times The PCR product was run on a 1% agarose gel with a 100 bp ladder and visualized with ethidium bromide As positive and negative... Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp strain PCC6803 II Sequence determination of the entire genome and assignment of potential protein-coding regions DNA Res 3, 109–136 24 Morgenstern R, Guthenberg C & DePierre JW (1982) Microsomal glutathione transferase Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione... harboured the plasmid pLys SL [60]) using the same protocol Glycerol stocks were prepared and stored frozen at )70 °C for subsequent use as starting material for the expression experiments Overexpression of SynMGST and E.coliMGST and preparation of membrane fraction Small aliquots (1–2 lL) of bacterial glycerol stock were grown in 2· YT medium overnight at 37 °C The cultures were diluted 1 : 100 into 2 L of. .. start site would be the in-frame ATG, 30 nucleotides downstream of the GTG This coding region was amplified using the sense primer 5¢-GAGAGACATATGGTAAGC GCGCTGTACGCC-3¢ PCR was performed with 0.2 mm dNTPs, 2 mm MgCl2, 0.25 lm of the respective primer, about 0.1 pmol of template and 0.5 U of Pfu polymerase The temperature cycles 1698 A Bresell et al were 30 s at 94 °C, 1 min at 40 °C and 2 min at 72 °C,... isolated from a number of clones and cleaved with NdeI and HindIII followed by agarose gel electrophoresis to verify the size of the inserts Selected inserts were sequenced on an Applied Biosystems (Foster City, CA, USA) 373A automated DNA sequencer using a dye terminator cycle sequencing kit The expression construct containing the correct coding sequence for both the SynMGST and E.coliMGST was transformed . Synechocystis sp. bacteria further, to gain insight into the function of these proteins and the evolution of the MAPEG superfamily. Cloning and overexpression demonstrated. Bioinformatic and enzymatic characterization of the MAPEG superfamily Anders Bresell 1, *, Rolf Weinander 2, *, Gerd Lundqvist 3 ,