* Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France
Microbiology, Free University of Brussels (VUB) and J. M. Wiame Research Institute1, Brussels, Belgium
Correspondence: E-mail: labedan{at}igmors.u-psud.fr.
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Abstract |
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Key Words: aspartate carbamoyltransferase catalytic PyrB subunit regulatory PyrI subunit dihydroorotase protein-protein interactions coevolution process linear correlation coefficient
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Introduction |
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First, the protein ATCase has been intensively studied over the last 40 years as a model of an allosteric enzyme (see Hervé 1989; Lipscomb 1994, for reviews), and crystal structures have been resolved at high resolution for several prokaryotic ATCases: those of Escherichia coli (Lipscomb 1994; Beernink et al. 1999 and references therein), Bacillus subtilis (Stevens, Reinich, and Lipscomb 1991), and Pyrococcus abyssi (Van Boxstael et al. 2003). All ATCases are composed of a basic subunitthe product of gene pyrBwhich assembles in catalytic homotrimers. The PyrB polypeptide itself is composed of two structural domains which respectively bind the substrates carbamoylphosphate (N-half) and aspartate (C-half). Several classes of ATCase holoenzymes are known according to their mode of association with other proteins (table 1).
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Eukaryotic ATCases also are of different types. In plants, the situation is similar to the prokaryotic class C, except that the enzyme is sensitive to allosteric effectors (Khan, Chowdhry, and Yon 1999; Williamson and Slocum 1994). In animals and in Dictyostelium discoideum, pyrB is fused to the genes for carbamoylphosphate synthetase (EC 6.3.5.5) and DHOase in a multifunctional unit encoding the so-called CAD protein (Coleman, Suttle, and Stark 1977; Davidson et al. 1993). The CAD native structure is a hexamer of identical subunits where the ATCase domain plays a central role in oligomer formation (Qiu and Davidson 2000). In Fungi, a CAD-like protein occurs, where the DHOase segment is not catalytically functional, reminiscent of prokaryotic subclass A2 (Souciet et al. 1989). In trypanosomes, a non-fused pyrB gene is present, clustered in an operon-like pattern with other pyr genes (Gao et al. 1999).
Our second main reason to focus on ATCase was based on a previous analysis of the evolutionary history of both aspartate-carbamoyltransferase and ornithine carbamoyltransferase (OTCases), a pair of paralogous enzymes present in nearly all organisms (Labedan et al. 1999). The phylogeny of 33 ATCases and 44 OTCases proved to diverge widely from the cognate SSU rRNA organismal tree because both gene trees turned out to be polyphyletic. This intricate topology could not be used to root the Tree of Life, but it could be rationalized in a rather simple scenario when we recognized that any ATCase belongs to one of two familiesATC I and ATC IIwhich could be traced back to gene duplications having occurred in the Last Common Ancestor (LCA) to all extant life or even before its emergence (Labedan et al. 1999). Likewise, present-day ornithine carbamoyltransferases belong to two ancient families, OTC and OTC ß (Labedan et al. 1999).
Because these families of carbamoyltransferases had been defined uniquely on phylogenetic grounds, we attempted to correlate these data with other properties of these enzymes. In this article, we propose a new approach which, when applied to the ATCase case, confirms our hypothetical model. In a first step, we show that the PyrB phylogeny corresponds closely with the different classes of quaternary structures of ATCases. We further show that evolution of ATCases has been shaped by the interaction of PyrB with various partners in the different holoenzymes. This coevolution process was assessed by estimating directly the degree of correlation between the phylogenetic trees for pairs of interacting proteins using a statistical analysis. This second major component of our present work is based on the method recently developed by Goh et al. (2000) and Pazos and Valencia (2001). In this step, we further stress how crucial it is to distinguish among speciation effects, structural interactions, and functional interactions. We anticipate that the approach described in this article could be useful to ascertain scenarios proposed to trace back the history of protein families displaying either polyphyletic trees or other uncertain phylogenies.
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Materials and Methods |
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Reconstructing Phylogenetic Trees
Rooted phylogenetic trees were derived from multiple alignments of sets of filtered ATCases and OTCases using two different approaches.
Assessing the Correlation Between Phylogenetic Trees of Interacting Proteins
As already proposed by Goh et al. (2000) and Pazos and Valencia (2001), the correlation between evolutionary trees was measured at the level of their respective distance matrices. Because the DARWIN approach is based on maximum likelihood (Gonnet, Cohen, and Benner 1992), we used the PhyloTree program to build matrices of both the PAM distances separating each sequence from all the others and their respective variances. A script was designed to collect automatically for each pair of interacting proteins belonging to the same set of species, their trees, and both matrices (PAM distances and variances of these PAM distances). Routinely, after checking that both evolutionary trees displayed correlated topologies, we directly estimated the Pearson's correlation coefficient r (Press et al. 2002) between the pairwise sequence distances. Calculation of r between the respective matrices was made using a Microsoft Excel 2000 automatic procedure.
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Results |
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Interestingly, the few ATC II bacteria that are deprived of pyrI are not clustered with the class B members. This is the case for the Spirochete Leptospira interrogans [PYRB_LEPIN] and for a small and monophyletic group of five bacterial genera (e.g., PYRB_BORPE), which branches far from the class B clade, inside the second main ATC II cluster containing all eukaryotes. In this cluster, all CAD (animals [e.g., PYR1_MOUSE] and D. discoideum [PYRB_DICDI]) and CAD-like (fungal) sequences (e.g., PYRB_YEAST) form a clade which had a close common ancestor with the Trypanosomatidae sequences ( [PYRB_LEIME], [PYRB_TRYCR]). This appears to be in good agreement with previous data (Gao et al. 1999), which suggested that Trypanosoma sequences are progenitors in the evolution to CAD. The plant sequences (e.g., [PYRB_ARATH]), which seem structurally analogous to class C enzymes, form another clade. The present topology of the ATC II family strongly suggests a common history of eukaryotic ATCases and prokaryotic B class ATCases, despite their different quaternary structures, and does not support previous models where eukaryotic ATCases were supposed to derive from prokaryotic A class (Schurr et al. 1995).
Figure 1 further shows that the ATC I family, made only of bacterial enzymes, contains all sequences belonging to classes A and C. The great majority of C enzymesto the notable exception of Xanthomonadales ([PYRB_XANCA], [PYRB_XYLFA])forms a clade grouping the low GC Gram-positive bacteria (e.g., [PYRB_BACSU]), Fusobacterium nucleatum [PYRB_FUSNU] and Aquifex aeolicus [PYRB_AQUAE]. We also observed tight clustering of the different known A enzymes. The A1 sequences appear to be monophyletic and group all high GC Gram-positive (e.g., [PYRB_MYCTU]) bacteria (except the Bifidobacteriales ([PYRB_BIFLO]), and Tropheryma [PYRB_TROWH]) and two members of the Thermus-Deinococcus branch (T. aquaticus [PYRB_THEAQ] and D. radiodurans [PYRB_DEIRA]). Known A2 sequences belong to a large cluster, where they are interspersed with some uncharacterized ATCases. In this cluster, the Cyanobacteria (e.g., [PYRB_THEEL]) are monophyletic and group together with a set of phylogenetically distant bacteria, such as members of the newly defined Bacteroidetes-Chlorobi group (Cytophaga hutchinsonii [PYRB_CYTHU], Chlorobium tepidum [PYRB_CHLTE]), Fibrobacteres (Fibrobacter succinogenes [PYRB_FIBSU]) and either magnetotactic (Magnetococcus [PYRB_MAGMC]) or delta (Desulfovibrio [PYRB_DESDE], Geobacter metallireducens [PYRB_GEOME]) proteobacteria. There is also a larger clade of A2 sequences grouping alpha (e.g., [PYRB_AGRTU]), beta (e.g., [PYRB_NITEU]), and gamma (e.g., [PYRB_PSEAE]) Proteobacteria. The fact that A and C ATCases appear together in the same family indicates that they descend from a common ancestor, differing by the fact that they are or are not stably associated with an active or inactive DHOase.
Correlation Between Phylogenetic Families of ATCases and of DHOases
Because a phylogenetic classification has been previously worked out for DHOases (Fields et al. 1999), we compared it with our ATCase data in order to obtain more information about the physical interaction of these two proteins at the holoenzyme level in ATCases of class A. Two main types of DHOases, I and II, defined according to their sizes, appear to derive from one ancestral protein of the amidohydrolase superfamily (Holm and Sander 1997), type I being the most ancient because it occurs in all three domains.
Several classes have been delineated within each type according to their mode of interaction with other pyrimidine enzymes (Fields et al. 1999). In the larger type I we find (1) a class a, made of non-interacting and active DHOases present in low GC Gram-positive bacteria and in archaea; (2) a class b, made of DHOases interacting with PyrB and either active, as in high GC Gram-positive bacteria and the Thermus-Deinococcus clade, or inactive as in Proteobacteria and Cyanobacteria; (3) a class c, made only of eukaryotes and subdivided in active DHOases present in animals and in inactive DHOases present in fungi; and (4) a class d, which groups a special category of poorly defined DHOases present essentially in a few Proteobacteria and Cyanobacteria. The organisms possessing an inactive DHOase of type I (Proteobacteria, Cyanobacteria, Fungi) also contain a smaller, active DHOase of type II. Plants also have a type II active DHOase which would have a bacterial endosymbiotic origin (Fields et al. 1999). Whether a DHOase-like protein is catalytically active or not can be tentatively inferred from the presence or absence of four catalytically critical histidine residues in the derived amino acid sequence (Fields et al. 1999).
Figure 2 and table 2 show the correlations between the families and classes of quaternary structures we have delineated for the prokaryotic ATCases and the different classes of DHOases (Fields et al. 1999). Figure 2 details for each ATC structural class the nature of the associated DHOase, and table 2 summarizes this rather complex situation. All species not belonging to class A2 contain a unique DHOase which is of type Iexcept in the case of the Proteobacteria belonging to class B. As expected, this unique DHOase is active and may belong to class a (accompanying an ATCase of class B or C), class d (with an ATCase of class B or C) or class b (with an ATCase of class A1). The organisms harboring an ATCase of class A2 present a more complex set of DHOases. Beside the Ib inactive form (PyrX), which is associated with the PyrB polypeptide at the quaternary level, there is another active DHOase which may apparently be either of type I or II. Moreover, some A2 organisms such as Pseudomonas may even have both active forms Id and IIa.
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Study of these ATCase-DHOase correlations may help in assigning a putative structural class to a few of the experimentally uncharacterized ATC I sequences. For example, the epsilon Proteobacteria Helicobacter pylori and Campylobacter jejuni contain two DHOases, a Ib inactive and a IIa active, and their pyrB and pyrX genes are distant. This is reminiscent of the Cyanobacteria and would suggest an A2 quaternary structure for these epsilon proteobacterial ATCases (PYRB_HELPY and PYRB_CAMJE in fig. 2).
Assessing the Coevolution of ATCase and of Its Partner Proteins in the Different Holoenzymes
The correlations observed above between phylogenetic clustering and classes of quaternary structures suggest the occurrence of some underlying process of gene coevolution that monitors interactions at the level of the quaternary structures of the proteins considered. To check this hypothesis, we tried to estimate the extent of this coevolution process using a recently published methodological approach (Goh et al. 2000; Pazos and Valencia 2001). This approach is based on the comparison of the phylogenetic trees obtained for a pair of intimately interacting proteins. Because comparison of phylogenetic trees could be not accurate enough, it has been proposed (Goh et al. 2000; Pazos and Valencia 2001) to compare directly the distance matrices of each interacting protein using the same set of species. The degree of correlation is estimated by determining the linear correlation coefficient r (Pearson's correlation coefficient calculated as in Press et al. 2002) between the pairwise sequence distances. It is generally admitted that a Pearson's correlation coefficient r below 0.3 means no association (Cohen 1988). An r value falling in the interval 0.30.7 indicates a weak association (Cohen 1988). The association is supposed to be strong if r is larger than 0.7 (Cohen 1988). Moreover, in the case of protein-protein interactions, Goh et al. (2000) and Pazos and Valencia (2001) agreed on a value of 0.79 as a threshold of significance for the linear correlation coefficient between the divergent evolution of each of the interacting proteins. Although some interacting proteins were found below this threshold and a few non-interacting proteins were above it (Goh et al. 2000; Pazos and Valencia 2001), it appeared that this value is the best compromise between sensitivity and specificity (see also Goh and Cohen 2002). Accordingly, we chose an empirical cut-off of 0.8 to ascertain if a positive correlation reflects such a coevolution process.
Table 3 and the accompanying figure 3 show how we tried to differentiate for each ATCase class the functional, structural, and functional/structural interactions taking place at the holoenzyme level. First, we have two caseswhich may be seen as internal negative controls in our experimental testwhere no structural interaction is expected, although a functional interaction is possible. In the case of the 23 species (16 genera) harboring an ATCase of class C, a Pearson's correlation coefficient of 0.613 was found between the distance matrices established for the PyrB and PyrC polypeptides, respectively. This relatively low value may correspond to some functional interaction between these two enzymes, which are involved in the two first steps of the pyrimidine biosynthesis pathway. Likewise, in the case of ATCase classes B and B' (49 species, 32 genera), the correlation between PyrB and the DHOase remains weak (r = 0.525). On the contrary, in the same B and B' classes, we find a high correlation (r = 0.857) corresponding to the known structural interaction occurring between PyrB and PyrI. A more complex case implying both structural and functional interactions between PyrB and the active DHOase of type Ib can be evaluated in the case of the set of species belonging to subclass A1. A significant correlation (r = 0.759) is obtained, but this Pearson's coefficient is slightly below the cut-off previously set (Goh et al. 2000; Pazos and Valencia 2001). This difference may be due to the small size of this group of sequences (only 11 species, 7 genera). Note, however, a rather high value for the correlation between the respective variances in the case of subclass A1. Finally, in the case of subclass A2, there is a clear-cut difference for the same set of 28 species (24 genera) between the r values calculated between either PyrB and its structural partner, the inactive DHOase of type Ib, (0.917), or the same PyrB polypeptide with the other, active, DHOase (0.250) with which it does not structurally interact.
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Discussion |
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Such a complex situation suggests an intricate evolutionary history that we have tried to partially disclose in this work. The precise structural and/or functional interaction of various polypeptides requires fine-tuning processes of coevolution of their encoding genes. To detect such processes of concerted evolution and to differentiate them from other mechanisms of parallel evolution, we used several complementary approaches aiming at discriminating between different underlying factors such as gene proximity and speciation.
In a first and major step of our experimental approach, we have disclosed a clear correlation between the different quaternary structures of ATCases and the two families ATC I and ATC II we had previously described solely on phylogenetic grounds (Labedan et al. 1999). As shown in figure 1 and summarized in table 2, all ATCases belonging to class B form a unique clade inside the ATC II family. All members of this clade, as well as the two species belonging to class B' are prokaryotes containing a pyrI gene. Thus, the mere presence of a pyrI gene in a newly studied organism would be a signature for a class B ATCase. This ATC II family contains a set of bacteria without a pyrI gene, and nothing is known about the ATCase quaternary structures of this set. The rest of this ATC II family is made up of all known eukaryotic ATCases. Thus, contrary to a previous proposition by Schurr et al. (1995), the genes encoding these eukaryotic ATCases do not appear to derive from a class A ATCase but probably have evolved in parallel with those encoding prokaryotic ATCases of class B.
Remarkably, the ATC I family contains all the ATCases of classes A and C, suggesting a common ancestry for these two very different classes of quaternary structures. The situation appears to be more complex than in the ATC II family, because neither class A nor class C is monophyletic. For example, a majority of the class C members forms a clade containing the low GC Gram-positive bacteria, Fusobacteria and Aquifex, but the Xanthomonadales are in a distant position. Note that the Xanthomonadales also differ from the other species harboring a class C by having an active DHOase of type Id and not Ia (fig. 2). It is likely that, while subclass A1 is monophyletic, subclass A2 is divided into two clades separated by several bacteria, for which we do not have experimental evidence about the quaternary structure of their ATCase, and by class C Xanthomonadales.
Moreover, the evolution of the ATC I family may have been influenced by the nature of the DHOase interacting with the ATCase in the two subclasses A1 and A2. This influence has been examined with different approaches. The conservation of gene proximity in phylogenetically distant species is classically interpreted (see, for example, Dandekar et al. 1998; Marcotte et al. 1999) as strong evidence for either functional or structural interaction of their products, or their common regulation. In classes B and C where there is at best a functional interaction between these two enzymes which share a common molecule (N-carbamoyl-L-aspartate) in the pyrimidine pathway, it seems irrelevant whether their encoding genes (pyrB and pyrC) are clustered or not. In class A, some strong pressure has kept these genes (pyrB and either pyrC or pyrX) adjacent, with the notable exception of the Cyanobacteria. Therefore, only the occurrence of gene proximitynot the absence of ithas any suggestive value in searches for interactions between gene products.
A much better way to test if genes have evolved in a concerted way because their encoding proteins are supposed to interact is to assess the degree of correlation between their phylogenetic trees. The requirement for structural interaction at the quaternary level has been measured by a statistical approach (Goh et al. 2000 ; Pazos and Valencia 2001). As summarized in table 3 and figure 3, the linear correlation coefficient (Pearson coefficient) between the pairwise sequence distances of PyrB and the other partner proteins was found to be larger than the threshold value of 0.8 (Goh et al. 2000; Goh and Cohen 2002; Pazos and Valencia 2001) in the case of PyrB interacting with either PyrI (0.857) or PyrX (0.917). The known structural interaction PyrB-PyrC in subclass A1 was found to be slightly below this threshold (0.759). This lower value may be explained in two ways: either by a sampling effect (only 11 species available) or, more interestingly, by a compromise between antagonistic forces corresponding to the dual role of these DHOases, which are catalytically active but also act as a structural partner of PyrB in the A1 species. Indeed, mutations of functional residues usually decrease the activity, but concurrently they often increase stability of the protein (Kirschner and Gerhart 1998). In the case of DHOases present in A1 species, their encoding genes must at the same time avoid any deleterious change in the residues that are necessary for the good functioning of its product as well as in those required for maintaining a 3D protein structure able to interact at the quaternary level with the PyrB partner. The equilibrium between these potentially antagonistic effects on the pyrC genes present in A1 species may be reached only at this lower level of correlation.
In conclusion: (1) We have disclosed a very strong correlation between the phylogeny of ATCases and the different classes of quaternary structures of this enzyme, suggesting unexpected common ancestry for prokaryotic B and eukaryotic ATCases on the one hand and for A and C on the other. (2) We have quantitatively assessed the structural constraints which underlie the interactions occurring between the partner proteins of these classes. The emergence of specific quaternary structures appears to have been a more recent event than the separation into the ATC I and ATC II families. As outlined at the beginning of this article, the phylogeny of carbamoyltransferases diverges widely from the organismal tree of life based on small-subunit rRNA. The correlations we have established strenghten the significance of this polyphyletic pattern, the origin of which will be discussed in a forthcoming article.
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Acknowledgements |
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This work was supported by the Flanders Foundation for Joint and Fundamental Research and by the Centre National de la Recherche Scientifique (CNRS) (UMR 8621). Daniil Naumoff was supported by a postdoctoral grant from the French Ministère de la Recherche.
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Footnotes |
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Michele Vendruscolo, Associate Editor
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