Modelling the evolution of the archaeal tryptophan synthase

Background: Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate. Results: In order to study the evolution of trp genes, completely sequenced archaeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o) contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operonbased trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i) consisted of trpB2 genes located inside, the other (trpB2_a) of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archaeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium. Conclusion: In archaeal genomes, several stages of trpB evolution, TrpA/TrpB cooperation, and operon formation can be observed. Thus, archaeal trp genes may serve as a model system for studying the evolution of protein-protein interactions and operon formation. Background The synthesis of tryptophan is a common metabolic capability of microorganisms and higher plants, which is not provided by mammals. The prokaryotic trp operon encodes the enzymes catalysing the final and pathwayspecific steps from chorismate to L-tryptophan. For more than 40 years, the enterobacterial operon has now been the classical model system for studying the evolutionary relation of genes and enzymes (see [1,2] and references therein) as well as gene regulation. Considering gene regPublished: 10 April 2007 BMC Evolutionary Biology 2007, 7:59 doi:10.1186/1471-2148-7-59 Received: 13 February 2007 Accepted: 10 April 2007 This article is available from: http://www.biomedcentral.com/1471-2148/7/59 © 2007 Merkl; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 20 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:59 http://www.biomedcentral.com/1471-2148/7/59 ulation, several, conceptually quite different mechanisms have been described for the trp operon. Most of them were elucidated in bacterial species (see e.g. [3-5], and references therein). However, regulation of trp operon expression has also been shown for the archaea Methanothermobacter thermoautotrophicus [6,7] and Thermococcus kodakaraensis [8]. The reason for an elaborated regulation may be the fact that tryptophan is one of the amino acids, whose biochemical synthesis is very expensive [9]. Besides regulation, other features of tryptophan biosynthesis have been studied extensively. The composition of the operon and several aspects of its evolution have been analysed [10], and for each enzyme, at least one 3D-structure has been determined. Taken together, the trp operon is besides the ribosomal protein operons one of the best-characterised gene clusters occurring in microorganisms. Its investigation has provided fundamental insights into many aspects of bacterial genetics and enzymology; see [2]. The canonical trp operon encodes seven enzymes responsible for the synthesis of L-tryptophan from chorismate. The first reaction is catalysed by the anthranilate synthase, a glutamine amidotransferase, which is a complex consisting of the larger synthase (TrpE) and a smaller glutaminase (TrpG) subunit. The anthranilate phosphoribosyl transferase (TrpD) provides the glutamine amidotransferase function that allows glutamine to serve as the amino donor in anthranilate formation. The two subsequent enzymes, TrpF and TrpC, catalyse the isomerisation of phosphoribosylanthranilate and the synthesis of indole-3-glycerol phosphate, respectively. TrpA and TrpB constitute the αββα tryptophan synthase complex which catalyses the final reaction from indole-3glycerole phosphate + L-serine to L-tryptophan + H2O. The α subunit (TrpA) cleaves indoleglycerol-3-phosphate to glyceraldehyde-3-phosphate and indole. The latter is transported through a hydrophobic tunnel to the associated β subunit (TrpB), where it is condensed with L-serine to yield L-tryptophan [11]. A sophisticated mechanism of allostery links the α and β monomers of the synthase; see e.g. [12]. Several Trp enzymes represent paradigmatically larger classes of proteins having similar function or protein architecture: TrpG is similar to HisH (an enzyme involved in histidine biosynthesis) and other glutaminases of type I glutamine amidotransferases [13]. TrpF, TrpC and TrpA are all (βα)8 barrels possessing similar phosphate binding sites [14]. The basic (βα)8 barrel is the most common enzyme fold in the PDB database of known protein structures [15]. For the bacterial trp genes, the following order was determined: large anthranilate synthase subunit (trpE), small anthranilate synthase subunit (trpG), anthranilate phosphoribosyl transferase (trpD), indole-3-glycerol phosphate synthase (trpC), phosphoribosyl anthranilate isomerase (trpF), tryptophan synthase β subunit (trpB) and tryptophan synthase α subunit (trpA), or abbreviated trpEGDCFBA [16]. The gene-fusions trpGD and trpEG have been observed in several species; moreover, in other genomes, the operon is broken up into several gene clusters. In archaeal genomes, order of trp genes is highly variable. In Sulfolobus solfataricus, an intact operon trpBADFEGC is observed. In Haloferax volcanii, the trp operon is divided into two isolated clusters, trpCBA and trpDFEG, separated by more than 1200 kb. In the genome of Natronomonas pharaonis, there exist three homologs of trpD and two homologs of trpB, trpE and trpG each. Pyrococcus horikoshii completely lacks the genes for tryptophan synthesis (and for other aromatic amino acids). The genes trpB, trpA and trpE, trpG are frequently in the same order and in close proximity, i.e. they comprise the linkage groups trpBA and trpEG. In both cases, the gene products constitute a bienzyme complex, whose active centres interact with each other. Because they occur in both bacterial and archaeal genomes, these linkage groups have been identified as ancestral [16]. A reconstruction of the tentative ancestral trp operon is hampered by the observation that trp genes are poor phylogenetic reporters. Different rates of evolution, multiple gene duplications and convergent evolution, as a consequence of specific adaptation to environmental demands, may be the reason for inconsistencies seen in comparisons of phylogenies deduced from trp genes or rRNA [16]. Therefore, the evolution of each element of the trp operon has to be examined separately. For evolutionary studies, tryptophan synthase is an especially interesting candidate. This enzyme has been analysed for decades in order to understand the structural basis and functional consequences of protein-protein interactions [17]. The isolated TrpA and TrpB proteins form stable, however poorly active α monomers and ββ homodimers, respectively [18,19]. Their assembly to the native αββα complex induces conformational changes in both subunit types, as shown by X-ray crystallography for the Pyrococcus furiosus synthase [18]. The result of this communication between the α and β subunits is a reciprocal activation by one to two orders of magnitude [20]. Conformational changes crucial for the allosteric communication between the active sites of the α– and β-subunits have been analysed in detail for the Salmonella typhimurium tryptophan synthase; see e.g. [21-24]. Page 2 of 20 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:59 http://www.biomedcentral.com/1471-2148/7/59 The role of the β-subunit is of particular importance for the evolution of Trp synthase. For archaea and bacteria, it is known that two variants of trpB genes occur, which can clearly be distinguished by their protein sequences [25]. The major group, harbouring proteins of type TrpB1 includes the enzymes of enterobacteria and Bacillus subtilis. The minor group (denoted TrpB2) contains many archaeal proteins. Most prokaryotes like E. coli possess a single trpB1 gene. However, in several bacterial and archaeal genomes, a combination of one trpB1 and one trpB2 gene occurs. In addition, some species exist, which have only one or two trpB2, but no trpB1 gene. This variety prompted us to characterise the evolution of TrpB and its interaction with TrpA in detail, both biochemically and in silico. Based on biochemical findings, a model for the evolution of the tryptophan synthase complex has recently been introduced [26]. This model assumes the existence of an ancient and non operon-based trpB2. After duplication, only one trpB2 gene presumably has been integrated into the trp operon. Differences in evolutionary pressure may have been responsible for the divergence of non operonand operon-based trpB genes. The coevolution with trpA may have led to a better adapted trpB1. The data on complex formation and subunit activation led us consider existing trpB variants as representatives of evolutionary steps in the postulated model. In this study, I have assessed this model by phylogenetic methods. Two basic questions have been addressed: i) What is the evolutionary relationship of trpB1 and trpB2? ii) How did extant archaeal trp operons evolve? Extending previous work [25], I will discuss novel hypotheses concerning the properties of TrpB2 and operon formation. Based on the content of 26 completely sequenced archaeal genomes, comparative analyses of trp sequences, and their locations in genomes will be reported in order to reconstruct the evolution of TrpB-type subunits and of the coevolution of TrpA/TrpB. It will be shown that TrpB2 variants represent different stages of TrpA/TrpB cooperation and that TrpB2 is favoured over TrpB1 in certain environments. Moreover, TrpB2 has features of a more ancient TrpB variant. Results and Discussion Assessing the composition of trp gene clusters In order to describe the composition of trp regulons in a quantitative manner and to compare their content in archaeal and bacterial genomes, AMIGOS [27] was used. By comparing genomes, this program identifies gene clusters and rates each individual cluster element with a consCL-score. The consCL-score of an individual gene depends on i) the occurrence of this gene in a given gene cluster and ii) the global similarity of the genomes harbouring these clusters. Thus, individual scores assess both the relatedness of genomes and the frequency with which individual genes are members of a cluster. The higher a score, the more pronounced is the occurrence of an individual gene in a given gene cluster. Table 1 lists consCLscores for elements of archaeal and bacterial trp operons. The numbers indicate that in bacteria the clustering of trpA and trpB1 was stronger than that of all other trp genes. In archaeal genomes, the clustering of trpE and trpG was most prominent. A reason for the lower score of trpB in archaeal trp operons was the occurrence of two trpB variants (trpB1 and trpB2) in these species. The scores signalled that trpB1 was more frequently part of an trp operon than trpB2. Moreover, the score for trpA was lower than that of trpE or trpG. It follows for archaea that trpA and trpB are less strictly integrated into trp operons than in bacteria. This suggests that either evolutionary pressure responsible for operon formation is less pronounced or that additional selective forces disfavour the integration of trpA and trpB into certain archaeal trp operons. It has been hypothesised that TrpB2 possesses a second function and acts as a serine deaminase [25]. This prediction has been deduced from the analysis of phyletic patterns, i.e. the absence of an encoded serine deaminase function in certain genomes. However, it has been shown that TrpB1 of Thermotoga maritima and TrpB2_o proteins of Sulfolobus solfataricus and T. maritima have poor serine deaminase activities [26]. An alternative method of nonhomologous gene annotation is the exploitation of gene neighbourhoods [28], as e.g. implemented with AMIGOS. For trpB2, AMIGOS did not detect a second conserved gene neighbourhood besides the one constituting trp operons. Thus, no clues for an additional function besides tryptophan synthesis have been deduced for trpB2 by this approach. A naming code for trpB genes The two variants of trpB occur in various genomes in different combinations [25]. In order to facilitate the analysis of phylogenetic trees, a naming scheme was introduced. Names of genes and gene products were generated according to the scheme SPECIES_LOC|TYPE|TAX. Here, SPECIES is an abbreviation of the species name (see Materials). LOC indicates the position of the specific trpB gene relative to a putative trp operon (more precisely: relative to a trpA gene). If two trpB genes occur in a genome, they were labelled _i (if the gene was located inside the trp operon) or _o (if located outside the operon). If only a single trpB gene occurred in the genome, it was labelled _s, if the gene was linked to trpA, and it was labelled _S, if it was separate from trpA. TYPE indicates the gene type. It is 1 for trpB1 and 2 for trpB2. Finally, TAX gives the taxonomical classification. It is C for Crenarchaeota, E for Euryarchaeota and B for Bacteria. The following examples Page 3 of 20 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:59 http://www.biomedcentral.com/1471-2148/7/59 explain how to resolve sequence names: Aperni_o2C was used to name a trpB gene in the genome of Aeropyrum pernix (Aperni), which occurred outside the trp operon (_o) and was of type trpB2 (2). As A. pernix is a Crenarchaeota, the name ends with a C. The _o notation indicates that a second trpB gene exists in A. pernix. This gene was consequently named Aperni_i2C, as it is a trpB2 gene inside the trp operon. Note that also pairs like Tmarit_i1B and Tmarit_o2B exist indicating the occurrence of a trpB1 gene inside and a trpB2 gene outside the trp operon. Sacido_s2C is the designation of a trpB2 gene located inside the trp operon. As Sulfolobus acidocaldarius possesses only one trpB gene, it was labelled with a _s. Since Thermoplasma volcanium possesses only one trpB gene, which is non operon-based and of type trpB2, this gene was named Tvolc_S2E. Designations of the encoded proteins were assigned in a corresponding way. Determining the occurrence of trpB genes In order to determine the distribution of trpB variants, the COG [29] and the STRING database [30] were used. For all completely sequenced archaeal and bacterial genomes, their occurrence was determined and their location was identified. Depending on the occurrence of trpB variants, archaeal species were grouped into five categories, named species-types in the following. Note that these speciestypes characterise the content of genomes. Links to the above naming scheme for genes are gene location and type. As Table 2 shows, there were six archaeal genomes possessing a single trpB gene of class trpB1 (s1 or S1 species), four genomes with a single trpB gene of class trpB2 (s2 or S2 species), five genomes harbouring one operon-based and one additional, non operon-based trpB2 each (i2_o2 species), ten species of type i1_o2 (one operon-based trpB1 and one additional trpB2 gene) and one species possessing one operon-based and at least one non operonbased trpB1 gene (i1_o1 species). The most frequent combination (10 out of 26) was an operon-based trpB1 and a non operon-based trpB2 gene (i1_o2 species). N. pharaonis was the only archaeal species of type i1_o1. All five completely sequenced Crenarchaeota possess exclusively genes of class trpB2. Bacterial species did not contribute species-types noticeably different from those observed among archaea (data not shown). Both Geobacter species represent special cases most plausibly explained by ongoing genomic rearrangements: Gsulfu_i2B is an operon-based trpB2 gene of type TrpB2_o. The trp operon of G. sulfurreducens harbours both a trpB1 and a trpB2 gene. According to the annotation, the trpB1 gene (Locus tag GSU2375) contains a frameshift and is annotated as a pseudogene [31]. A direct neighbour of trpB1 in G. metallireducens is a transposase, making a recent transfer of this gene plausible. In comparison to archaea, the occurrence of trpB2 was less frequent in bacterial genomes and none contained exclusively trpB2 genes. Assessing phylogenetic relationship of trp genes Sequences originating from all archaea and several representative bacteria were selected for a phylogenetic classification of trp genes. Multiple sequence alignments were created by using M-Coffee [32], and trees were constructed and evaluated using SplitsTrees [33]. Figures 1, 2, 3, 4 are plots of unrooted trees generated for protein sequences of TrpA, TrpB, TrpD, TrpE, and TrpG. In order to assess the statistical strength of individual edges, bootstrap resampling was used. For relevant edges, bootstrap values were plotted; see Figures 1, 2, 3, 4. The trees were analysed in detail, as follows. TrpB In agreement with previous findings [25], TrpB1 and TrpB2 clearly fall into two distinct groups. This distinction Table 1: conscl scores for trp genes consCl – values Protein COG # Function