1. Introduction
Glutathione (GSH, reduced form) metabolism is considered as ancient as the history of life, dating back to the evolution of the oxygen-containing atmosphere [
1]. Several defence mechanisms have evolved to remove the oxidizing agents that allow the organisms to adapt and survive, of which the enzyme Glutathione S-Transferase (GST)-mediated reaction plays a pivotal role in cellular detoxification of harmful xenobiotics and endobiotics [
2]. GSH and GSTs are believed to have evolved in response to the increase in oxygen in order to scavenge the generated reactive oxygen species [
3].
Generally, detoxification enzymes act as a first line of defence against the environment characterized by the presence of toxins and pollutants. The detoxification process is performed in three phases, phase I enzyme oxidation, reduction or hydrolysis of the substrate, introducing a reactive group that can be attacked by phase II enzymes, which conjugate the active substrate with small molecules making the conjugate more water-soluble for excretion from cells by phase III enzymes [
4]. Among these key enzymes, GSTs represent an integral part of the phase II step and detoxify xenobiotics through catalysing the nucleophilic attack by GSH on electrophilic carbon, sulphur, or nitrogen atoms of nonpolar xenobiotic substrates, thereby preventing their interaction with crucial cellular proteins and nucleic acids. This generates water-soluble glutathione conjugates linked by a thioether bond (GS-R) that can then be degraded, or excreted out of the cell [
5,
6].
GSTs are the largest superfamily of proteins found almost in all eukaryotes and restricted to certain prokaryotic bacteria including cyanobacteria, proteobacteria, phototrophs and a few Gram-positive bacteria [
7]. GSTs are divided into three different families: (i) cytosolic GSTs, (ii) mitochondrial GSTs, and (iii) microsomal (membranous) GSTs designated as MAPEGs (a membrane-associated protein involved in ecosanoid and glutathione metabolism) [
4]. Cytosolic GSTs, which constitute the largest GST family are currently recognized as alpha, beta, delta, epsilon, zeta, theta, lambda, mu, nu, pi, sigma, tau, phi, and omega [
4,
8]. Among cytosolic GST families, members of the same class possess sequence identity greater than 40%, whereas 20% identity was found between classes [
1]. The mitochondrial GSTs are an evolutionary distant form of cytosolic GSTs that constitute different topology and subcellular localization [
9]. Distant classes of GSTs identified in bacteria are nu, zeta, and eta [
10,
11] and in cyanobacteria are chi [
12], and rho [
13].
The structural features of GSTs are composed of two ligand binding sites, the first G-site specific for GSH, which possess a conserved group of amino acid residues in the amino-terminal domain and the second is the substrate binding H-site in the carboxyl-terminal domain, which is structurally much more variable among GST classes [
4]. The functional properties of amino acid residues forming the G-site are highly conserved among the different classes of GSTs, while the binding pocket of the H-site varies considerably among GST classes. This diversity governs the electrophile substrate specificity, ability to pick, and catalyse a large number of structurally variable substrates [
14].
Cyanobacteria are unique prokaryotes, which exhibit oxygenic photosynthesis and produce reactive oxygen species (ROS) as a result of photosynthesis. GSH and the GSH-dependent GST enzymes evolved concurrently to protect themselves against the ROS which they massively produce by their active photosynthesis. These cyanobacteria were reported to possess high concentrations of GSH in their cytosol and might have been the very first organism to harbour GSH-utilizing enzyme GST [
15]. Cyanobacteria are highly diverse with a comprehensive set of different morphotypes from primordial unicellular form to multicellular heterotrichous form with genomic DNA ranging from ~1.2 to ~12 MB [
16]. These large diversities may have a direct impact on the distribution and evolution of cyanobacterial proteins including GSTs. However, cyanobacterial GSTs have been poorly studied.
Though, cyanobacteria may be very well the first organism to have harboured GSH utilizing enzyme, ironically its GST has not been studied extensively so far. The puzzle of the evolution of the cyanobacterial GSTs and the lines between the groups to date is incomplete. Considering this fact, an attempt was made in this study, through genome-wide analysis and by examining the multiple characteristics such as distribution, motif or signature patterns available in the sequences using the phylogenetic approach. Nevertheless, details about the GST super-families in cyanobacteria are essential to gain deeper insight information about the GSTs versatility in detoxifying the diverse compounds. For this purpose, an extensive study was carried out to seek the complex pattern, divergence, and distribution of GSTs in the cyanobacterial orders.
3. Results and Discussion
Many prokaryotic and eukaryotic GST proteins are classified into a collection of family-like classes mainly based on the sequence, structural similarities, and differences in the organization and composition of their active sites [
28]. However, their distribution in each organism remains controversial. Cyanobacteria, a group of primordial prokaryotes have vast diversity in genome organization that serves as a major reason for the divergence of GST protein among the orders and make them promising targets for evolutionary analyses. GST sequences that possess less than 25% sequence similarity are classified as a different type and GSTs that share more than 40% sequence similarity are considered as the same class [
4,
29]. Considering the present scenario, we analysed the global distribution and phylogenetic relationship of GSTs among the cyanobacterial orders. Primary grouping of GSTs was performed based on the protein sequences and their degree of identity. The major criteria that support the classification and discrimination of GSTs among the cyanobacterial orders are as follows:
- I.
Presence of catalytically essential residues such as serine, tyrosine, and cysteine at the N- terminus [
28]. The three catalytic residues present in the N-terminus of GST are broadly found in all types along with the GST specific motifs that contribute a polar functional group to the glutathione (G) binding site [
30]. Changes in these signature residues result in functional variation, such as differences in catalytic properties, selective dimer formation, and substrate bindings.
- II.
Presence of the hydrophobic “lock and key” motif, the SNAIL/TRAIL motifs at the N-terminal end and presence of catalytic signature residue in C-terminal domain [
23].
- III.
Difference in the organization and composition of the active site [
31].
3.1. Cyanobacteria Contain Many Diverse Members of GSTs
Phylogenetic analysis of 322 GST sequences retrieved from 126 cyanobacteria [
17] showed a total of 10 GST clades (one clade with ‘chi’ GST class and the remaining nine clades with 11 new GST classes) The most primordial unicellular form of cyanobacterial order
Pleurocapsales was found in four GST clades, lower unicellular order
Chroococcales presence was broadly observed in eight distinctly separated clades, middle filamentous order
Oscillatoriales was found in seven conserved clades. Interestingly, higher heterocyst containing orders
Nostocales and
Stigonematales were found respectively in nine and five different clades (
Figure 1). The distribution of common and order specific GST show the hierarchy of GST evolution among the cyanobacterial orders.
Since this was the first comprehensive data on cyanobacterial GST, for a clear understanding a naming convention was given to cyanobacterial GST for each class with reference to the previous names of prevailing GST. The nomenclature of cyanobacterial GST was framed using the lower case “cy” for cyanobacteria preceding the GST followed by the upper case denoting as “X”, then Arabic numeral denoting sub-types (1, 2, 3...) (cyGSTX1-cyGSTX11) (
Table 1). The identified types which shared close similarities with already existing groups, were identified as “Chi” class (colour code-red). GSTs which possess partial similarity with other prokaryotic GSTs and specific forms standing as independent lineages were denoted as (cyGSTX1-cyGSTX11) (different colour code given to each type) (
Table 1). Overall, the order
Nostocales was the largest with nine different GST classes, followed by orders
Chroococcales, Oscillatoriales, Stigonematales, and
Pleurocapsales respectively with 8, 7, 5, and 4 classes (
Table 1). This phylogenetic analysis of cyanobacterial GSTs suggests a gene duplication event, which led to the independent evolution of different GSTs type among the cyanobacterial orders.
3.2. Cyanobacteria Type Discrimination Based on Conserved Features
Putative 322 GST sequences retrieved from 126 cyanobacteria were subjected to alignment which showed the conservation of 12 motifs (M) namely M1-SGAIL, M2-SGAIL (2), M3-SLAIL, M4-SNAIL, M5-STEIA, M6-SDDII, M7-SAEII, M8-SSAIA, M9-SAIIN, M10-SLEII, M11-SAVIN, M12-SKDIL with the architecture of “ES [GLNTARS][ADE]I[LAI]” (
Table 2) (
Supplementary Figures S1,S2). The serine present in the identified motifs was found to be conserved in all identified GSTs. However, other amino acids in the motif, not directly involved in GSH binding, were highly diverse within the order (
Table 2). This architecture was equivalent to SNAIL/TRAIL motif that is present in most GST classes that contribute polar functional groups to the GSH binding site [
28].
Similarly, a putative SNAIL/TRAIL-like motif (SGAIV) at amino acid positions 86–90 was reported in the sequence of
AtuGSTH1-1. The hydroxyl group of serine in motif SGAIV at position 86 forms a hydrogen bond with the γ-Glu portion of GSH, whereas the other residues of the motif are not directly involved in GSH binding [
23]. Likewise, in cyanobacterial GSTs, serine in the motif architecture was found to be highly conserved in all the GST classes, but an amino acid change was observed within the motifs which is shown in
Table 2. The N-terminal motif is an excellent target for discriminating the GST groups, because it is better conserved than others which possess the important part of the active site. However, it can further be powered by combining the presence of specific conserved motifs at the C-terminal end. The signature motif, found at the C-terminal end used for discriminating the cyanobacteria GST, is more degenerative.
Chi class reported as cyanobacteria specific is found in all orders possessing a unique motif SGAIL. Both Chi and cyGSTX1 GST groups share the same motif SGAIL (
Table 2) and hence, the differentiation between Chi and cyGSTX1 is further refined by the presence of specific signature sequences at the N- and C-terminal ends. The two signature motifs in Chi group are GG[PA][KR]SRAS and NPFGK[VL]P[VA]L that are replaced by ISPN[SGN]RIP and BADIA[TC]YP in cyGSTX1. These motifs serve as an important feature for the discrimination of cyanobacterial GSTs of Chi from cyGSTX1 type. S(FL)AI(LM) and SNA(IVM)(LM) motifs are found in cyGSTX2 and cyGSTX3 groups, where the signature residue L is replaced by N in the cyGSTX3 motif. However, cyGSTX4 has the entirely unique motif ST(EDA)IA (
Table 2). Likewise, cyGSTX5 possess the SD(DRV)I(IL) motif and cyGSTX6, cyGSTX7, and cyGSTX9 found in
Chroococcales, Oscillatoriales, and
Pleurocapsales possess SA(DE)II, S(ST)AI(AC), and SL(ED)I(IM) motifs respectively (
Table 2). The similarities in the motifs indicate the close functional relatedness and the ladder of the GST evolution among the cyanobacterial orders.
Nostocales, one of the evolved forms of cyanobacteria has been proved to have greater evolutionary divergence [
32]. Three order specific cyGSTX8, cyGSTX10, and cyGSTX11 found in the higher order
Nostocales have SAI(IV)N, SA(IV)IN, and SKDIL motifs which remain entirely specific from the other GST group’s motif. The vast change in the motifs indicates the functional divergence of the GST with relevance to the variable substrates. Further, this identified motif and signature are group specific, which can be used as a query on the BLAST server for the identification of specific cyanobacterial GST groups (
Supplementary Figure S2).
3.3. Evolutionary Divergence of Cyanobacterial GST
Phylogenetic analysis of 322 cyanobacterial GST for 126 cyanobacteria revealed five major clades and four minor clades (
Figure 1). GST type Chi, cyGSTX1, cyGSTX2, and cyGSTX3, form separate major clades 1,2, 3, and 4 containing genus of the five orders except
Pleurocapsales, indicating the absence of evolved forms of GSTs in primordial forms of cyanobacteria. cyGSTX4 in clade 10 is the only form of GST which is found in five orders of cyanobacteria. The hierarchal evolution of GST among cyanobacterial orders was evidenced by clade 8 (cyGSTX9) containing only the genus of primordial
Pleurocapsales representing as an ancestor of all GSTs followed by clade 9 (cyGSTX6) which possesses the genus of primordial
Pleurocapsales, lower order
Chroococcales, and middle order
Oscillatoriales. Clade 7 (cyGSTX7) contains only the genus of lower order
Chroococcales and middle order
Oscillatoriales, indicating this group has lost in primordial order
Pleurocapsales. Clade 6 contains a cyGSTX5 representing the genus of
Pleurocapsales, Chroococcales and
Nostocales which suggests that this type might be lost in middle order
Oscillatoriales. Clade 5 which is diverged into three minor clades cyGSTX8, cyGSTX10, and cyGSTX11 representing only the genus of
Nostocales, diverged only in higher orders, which is lost in the lower, middle, and primordial orders. This proves that the GST evolved hierarchy from primordial
Pleurocapsales to higher
Nostocales substantiated the functional diversification of GSTs among the cyanobacteria forms.
GST families have diverse substrate specificity, which was acquired by the selection pressure during the course of evolution. Phylogenetic analysis revealed varying clustering of GST among the cyanobacterial orders (
Figure 1). The distribution of GST among the cyanobacteria increases gradually from primordial
Pleurocapsales to evolved
Nostocales which clearly hints that the GSTs have undergone a series of selection pressure to craft a functionally divergent enzyme with high adaptability and significance. The average evolutionary distance among the five orders decreases from the lower unicellular forms to higher heterocystous forms which are 1.49 and 1.19 respectively (
Table 3) suggesting that the primordial
Pleurocapsales experienced a differential selection pressure which shaped the evolution of highly effective GSTs in higher order
Nostocales. The low percentage of evolutionary divergence reducing from primordial order
Pleurocapsales to higher order
Nostocales clearly evidenced the conserved nature of active residues.
3.4. Classification of GST Based on N-Terminal End, the G-Site
As the cyanobacterial GST sequences possess high sequence divergence, the identified 12 different GST classes were further analysed by the second level of grouping based on the distribution of the conserved signature residues present in the N-terminal end. The N-terminal end otherwise named as G-site adopts a thioredoxin like fold responsible for GSH substrate binding by providing a main chain donor and acceptor for GSH [
28].
The two catalytic centres in the GST, GSH substrate binding site (G-site) and the hydrophobic substrate binding site (H-site) contribute a major role in assorting the classes. The G-site responsible for primary function is conserved among all classes. The H-site responsible for scavenging the variety of xenobiotic substrate facilitates functional diversification, hence it is less conserved. Catalytic residues at the G-site contribute major amino acids at active site, based on the amino acid participation in GSH binding, GST is sorted into two major classes, a Y-GST group which uses tyrosine and S/C-GST group which uses serine/cysteine to activate the GST at the N-terminal end [
31].
The structural backbone of Y-type and S/C type are found to be similar but the change in conserved residue at specific position within the fold has an important implication in the catalytic mechanism. The S/C type GST which uses its representative serine or cysteine residue is positioned at the amino terminus of the helix 1 to activate the bound GSH and serves as key to catalysis in most studied eukaryote and prokaryote enzymes [
33]. Cyanobacterial GST X5, X6, X7, X9, and X11 have cysteine 26 or 74/serine 8 at the helix 1, but in some groups serine plays a critical function, despite the cysteine which is not required for the catalysis. Both cysteine and serine form a disulphide bond with GSH and transform the substrate. Some types have threonine in the position of S/C-GST and the threonine hydroxyl group forms a hydrogen bond with the sulfhydryl group of GSH. Cyanobacterial GSTX5, GSTX7, and GSTX11 have a reactive active residue serine which is replaced by cysteine in GSTX6 and GSTX9. The Y-type GST has tyrosine positioned at place 5 or 7 in the beta strand 1, the hydroxyl group in tyrosine serves as a hydrogen bond donor to the sulfur of GSH. The occurrence of active residue tyrosine at the beta strand 1 in the GST Chi, X1, X2, X3, X4, X8, and X10 designates it as Y-type (
Figure 2). The most ancestral form of GST uses cysteine to activate the GSH, which change to serine in the middle-evolved forms and successful changes to tyrosine for catalysis [
28]. Mapping of cyanobacterial GST onto the network leads to a new observation that has not been reported previously which also corroborated well with the phylogenetic tree. Sequence similarity poses strong evidence on separation of Y-type and S/C type of GSTs. The network fairly clusters all the GSTs in one, because it adopts a thioredoxin fold as a common domain.
Figure 3 emphasizes the network drawn at the threshold e-value of 1 × 10
−10, the network shows the multiple distribution of GSTs among the cyanobacteria. The distribution of the protein confirmed that Y type GSTs namely Chi, X1, X2, X3, X8, and X10 have grouped as a cluster, where C-type GSTs formed as a separate cluster. X9 and X6 are C-type GST that stood as a separate cluster. Likewise, the clustering of X5 and X11 belonging to S-type GST indicates the similarities, X4 is the only GST found in all orders clustered with X7 GST. These groupings represent the broad range of structural and functional diversity among the cyanobacteria. S-type GST is found in all orders of cyanobacteria, unlike C-type GST which was found in the lower and middle but absent in higher orders. Whereas Y-type GST is predominantly found in higher order
Nostocales, followed by
Stigonematales,
Chroococcales, and Oscillatoriales suggesting the divergence of GST among the orders.
This grouping shares the perfect correspondence with the phylogeny. Among the cyanobacterial orders, primordial Pleurocapsales is generally considered to be the most ancestral has three S/C-group and one Y-group GSTs. Subsequently, three and two S/C-group GSTs were respectively found in middle evolved forms Chroococcales and Oscillatoriales along with five Y-groups GSTs in common, and the later evolved Nostocales having seven Y-groups and two S/C-groups GSTs. Similarly, Stigonematales possess only five Y-group GSTs. The largest diversity was found in Nostocales, and some of the classes were found to be lost in the other orders, because they became non-essential or evolution shaped them into more specific groups.
3.5. Structural Comparison of Cyanobacterial GST G-Site
Representative GST structures cyGSTX1, cyGSTX6, and cyGSTX7 which belong to the Y-group and S/C-group were analysed. Despite the high sequence differences, the backbone structure remains the same with two distinct domains: G-site at the N-terminal end and H-site at the C-terminal end. The N-terminal end adopts a topology similar to thioredoxin folds with βαβαββα
x structural arrangements and also has an alternative βαβα linked to the C-terminal end through the α-helices. Atkinson and Babbitt [
31] reported the importance of tyrosine in Y-type and serine/cysteine in S/C- type and its location, implications for specificity differences between the classes of each subgroup. The sequence and structural analysis of all cyanobacterial GST types show that residues are found in two different locations. In Y-type, tyrosine is found in the β-strand 1, whereas in S/C- type the residue is found in the loop region before the α-helixes 1 at the N-terminal end. The degree of glutathione binding among these groups varies significantly which regulates the binding of substrate.
cyGSTX1 which belongs to the Y-group has the active site positioned among the cleft of the β- strands. GSH forms a non-covalent interaction with Tyr5, Ile29, Ile31, Gln40, Lys45, Il24, Phe56 which are located in the β-strands. The Tyr5 in β1 binds with the SH group of GSH, whereas Ile29, Ile31 from β2 form a hydrogen bond with the amine group of glutamine. Phe56 from β3 positions the GSH in the active site through Van der Waals interactions. Interaction of the OH group of Tyr5 not only activates the GSH, it also contributes to the stabilization by its interaction with α3 (
Figure 4). The catalytic activity of active residue tyrosine investigated with site directed mutagenesis showed severe impairment in glutathione binding when replaced with His, Val or Thr [
34]. A change in the topology of the active site will destabilize the interaction of GSH with GST resulting in different catalytic properties and signifying the importance of tyrosine in the Y-type GSTs.
Differently, in the S/C type the active site is shifted in between the helix which is entirely different from Y-type. cyGSTX7, belonging to S-type, positions the GSH between the α-helix 1, 4, and 8. Ser66 which is found in α-helix 1 interacts with the SH group of GSH, the interaction is further stabilized by α-helix 4 and 8, which forms an N-terminal domains (
Figure 5a). Caccuri et al. [
35] reported in
Lucilia cuprina GST, the mutation of Ser to Ala has less than 0.5% of normal activity which shows weak affinity towards glutathione. Similarly, in cyGSTX6 a C-type group, GSH is found in Trx domain, Cys12 present in α-helix 1 interacts with the SH group of GSH. The interaction is stabilized by the involvement of α-helix 6 and 7 (
Figure 5b). In human GST P Cys14 is reported to participate in the catalytic reaction of GST by stabilizing the conformation of the active-site loop, but not in the GSH, binding directly [
36]. Involvement of α-helix in GSH binding remains the same for both S and C type GST groups, but the active site shift was observed in the Y-type group indicating the ubiquity and diversity of GSTs among the cyanobacterial orders. The commonalities among the cyanobacterial GST groups clearly indicate that GST has evolved by means of convergent evolution, the thioredoxin folds provide the foundation for all forms of GSTs. The active site shift occurring in the recently evolved Y-type might enhance the catalytic activities with significance for evolutionarily conserved Tyr7.
4. Conclusions
It was observed that cyanobacterial GST, an important class of detoxification enzyme, has evolved in hierarchy and might possess a divergent functional role. This study highlights the distribution of the 12 GST classes among the cyanobacteria such as primordial order Pleurocapsales with four GST isoforms which serves as an ancestor, Chroococcales with eight classes in lower order, seven in middle order Oscillatoriales, five in Stigonematales, and nine in higher order Nostocales. Despite its high diversity, the evolutionary distance evidenced the retaining of the ancestral GSTs function in all cyanobacterial orders. All cyanobacterial GSTs having the backbone of thioredoxin fold are categorized into two groups namely Y-GST and S/C-GSTs. Pleurocapsales with a simple photosynthetic apparatus possess only four GST classes which are one Y-type and three S/C-type GST. The well-organized Chroococcales and filamentous forms Oscillatoriales have eight and seven classes of GST comprising five Y-type GST, three S-type, and two C-type GST groups. While, the order Stigonematales has five GST classes all comprising the Y-type GST group. On the other hand, heterocystous Nostocales possesses nine classes grouped into seven Y-type and two S-type GST groups. The shifted active site of Y-GST away from α1 might enhance the catalytic capabilities compared to S/C-GSTs. The distinctive characteristics of analysed GSTs evidence that recently evolved order Nostocales with higher Y-GST classes might possess more active GSTs which are more evolutionary divergent compared to the lower and middle order cyanobacteria.