Gene Descent, Duplication, and Horizontal Transfer in the Evolution of Glutamyl- and Glutaminyl-tRNA Synthetases

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA^Gln is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670–8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS—a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the ``crown' taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA^Gln and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria γ/β subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea–eukaryote divergence.     

Molecular Evolution of Nitrogen Fixation: The Evolutionary History of the nifD, nifK, nifE, and nifN Genes

The pairs of nitrogen fixation genes nifDK and nifEN encode for the α and β subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed. Received: 21 June 1999 / Accepted: 1 March 2000     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

The Root of the Universal Tree of Life Inferred from Anciently Duplicated Genes Encoding Components of the Protein-Targeting Machinery

The key protein of the signal recognition particle (termed SRP54 for Eucarya and Ffh for Bacteria) and the protein (termed SRα for Eucarya and Ftsy for bacteria) involved in the recognition and binding of the ribosome SRP nascent polypeptide complex are the products of an ancient gene duplication that appears to predate the divergence of all extant taxa. The paralogy of the genes encoding the two proteins (both of which are GTP triphosphatases) is argued by obvious sequence similarities between the N-terminal half of SRP54(Ffh) and the C-terminal half of SRα(Ftsy). This enables a universal phylogeny based on either protein to be rooted using the second protein as an outgroup. Phylogenetic trees inferred by various methods from an alignment (220 amino acid positions) of the shared SRP54(Ffh) and SRα(Ftsy) regions generate two reciprocally rooted universal trees corresponding to the two genes. The root of both trees is firmly positioned between Bacteria and Archaea/Eucarya, thus providing strong support for the notion (Iwabe et al. 1989; Gogarten et al. 1989) that the first bifurcation in the tree of life separated the lineage leading to Bacteria from a common ancestor to Archaea and Eucarya. None of the gene trees inferred from the two paralogues support a paraphyletic Archaea with the crenarchaeota as a sister group to Eucarya. Received: 19 March 1998 / Accepted: 5 June 1998     

Close Evolutionary Relatedness of α-Amylases from Archaea and Plants

The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58 residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)₈-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one based on the alignment of the substantial continuous part of the (α/β)₈-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes, which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases. This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases, especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya, Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence regions may indeed constitute the ``sequence fingerprints' of a given α-amylase. Received: 3 June 1998 / Accepted: 20 August 1998     

Dynamics and Phylogenetic Implications of MtDNA Control Region Sequences in New World Jays (Aves: Corvidae)

To study the evolution of mtDNA and the intergeneric relationships of New World Jays (Aves: Corvidae), we sequenced the entire mitochondrial DNA control region (CR) from 21 species representing all genera of New World jays, an Old World jay, crows, and a magpie. Using maximum likelihood methods, we found that both the transition/transversion ratio (κ) and among site rate variation (α) were higher in flanking domains I and II than in the conserved central domain and that the frequency of indels was highest in domain II. Estimates of κ and α were much more influenced by the density of taxon sampling than by alternative optimal tree topologies. We implemented a successive approximation method incorporating these parameters into phylogenetic analysis. In addition we compared our study in detail to a previous study using cytochrome b and morphology to examine the effect of taxon sampling, evolutionary rates of genes, and combined data on tree resolution. We found that the particular weighting scheme used had no effect on tree topology and little effect on tree robustness. Taxon sampling had a significant effect on tree robustness but little effect on the topology of the best tree. The CR data set differed nonsignificantly from the tree derived from the cytochrome b/morphological data set primarily in the placement of the genus Gymnorhinus, which is near the base of the CR tree. However, contrary to conventional taxonomy, the CR data set suggested that blue and black jays (Cyanocorax sensu lato) might be paraphyletic and that the brown jay Psilorhinus (=Cyanocorax) morio is the sister group to magpie jays (Calocitta), a phylogenetic hypothesis that is likely as parsimonious with regard to nonmolecular characters as monophyly of Cyanocorax. The CR tree also suggests that the common ancestor of NWJs was likely a cooperative breeder. Consistent with recent systematic theory, our data suggest that DNA sequences with high substitution rates such as the CR may nonetheless be useful in reconstructing relatively deep phylogenetic nodes in avian groups. Received: 10 November 1999 / Accepted: 16 March 2000     

Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into α^A and α^D types, which are present in all reptiles except crocodiles, where only α^A chains are expressed. The occurrence of the α^D chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate α^A and α^D chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (α^A) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved ∼3.5 (α^A) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times. Received: 15 September 1997 / Accepted: 4 February 1998     

On the Origin of Operons and Their Possible Role in Evolution Toward Thermophily

We present a hypothesis suggesting that close linkage of functionally related anabolic genes and their ultimate integration into operons developed under selective pressure as a molecular strategy which contributed to the viability of ancestral thermophilic cells. Cotranslation of functionally related proteins is viewed as having facilitated the formation of multienzyme complexes channeling thermolabile substrates and the mutual stabilization of inherently thermolabile proteins. In this perspective, the evolutionary scheme considered the most probable is the evolution of both Bacteria and Archaea by thermoreduction (Forterre 1995) from a mesophilic, protoeukaryotic last common ancestor (LCA) endowed with appreciable genetic redundancy.     

Gene structure of a chlorophyll a/c-binding protein from a brown alga: Presence of an intron and phylogenetic implications

A Laminaria saccharina genomic library in the phage EMBL 4 was used to isolate and sequence a full-length gene encoding a fucoxanthin-chlorophyll a/c-binding protein. Contrary to diatom homologues, the coding sequence is interrupted by an intron of about 900 bp which is located in the middle of the transit peptide. The deduced amino acid sequence of the mature protein is very similar to those of related proteins from Macrocystis pyrifera (Laminariales) and, to a lesser extent, to those from diatoms and Chrysophyceae. Seven of the eight putative chlorophyll-binding amino acids determined in green plants are also present. Alignments of different sequences related to the light-harvesting proteins (LHC) demonstrate a structural similarity among the three transmembrane helices and suggest a unique ancestral helix preceded by two β-turns. The β-turns are conserved in front of the second helices of the chlorophyll a/c proteins more so than in chlorophyll a/b proteins. Phylogenetic trees generated from sequence data indicate that fucoxanthin-chlorophyll-binding proteins diverged prior to the separation of photosystem I and photosystem II LHC genes of green plants. Among the fucoxanthin-containing algae, LHC I or II families could not be distinguished at this time. Received: 14 February 1996 / Accepted: 4 April 1996     

Identifying a Folding Nucleus for the Lysozyme/α-Lactalbumin Family from Sequence Conservation Clusters

Four subfamilies of c-type lysozyme and one subfamily of α-lactalbumin are defined from 78 sequences, and their folding nucleus is identified with a method based on conserved residues and native structural contacts between pairs of conserved residues. One large cluster of 19 conserved residues is found which is mostly nonpolar, buried, and nonfunctional. It can be subdivided into three subclusters: (1) conserved residues in four helices; (2) conserved residues that stabilize the connector between the α and the β domains; and (3) a β-turn, sitting in the middle of a bowl of α-helix residues. It is proposed that this folding nucleus initiates four helices, A, B, C, and D, three β sheets, and the connector, which corresponds closely to the nucleation of the so-called fast folding track pathway. As the secondary structures propagate, nonconserved residues and functionally conserved residues would form additional contacts. The conserved residues are selected with a phylogenetic scheme in which single members of subfamilies are selected. Subfamilies are then equally weighted to obtain the consensus conservation. Received: 11 June 2001 / Accepted: 28 August 2001     

The Last Universal Common Ancestor (LUCA) and the Ancestors of Archaea and Bacteria were Progenotes

The tRNA split genes of Nanoarchaeum equitans and the Met-tRNA^fMet → fMet-tRNA^fMet pathway, identifiable as ancestral traits, and the late appearance of DNA are used to understand the evolutionary stage at which the progenote → genote transition took place. The arguments are such as to impose that not only was the last universal common ancestor (LUCA) a progenote, but the ancestors of Archaea and Bacteria were too. Therefore, the progenote → genote transition took place in a very advanced stage of the evolution of the tree of life, and only when the ancestors of Archaea and Bacteria were already defined. These conclusions are in disagreement with commonly held beliefs.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Determining the Relative Rates of Change for Prokaryotic and Eukaryotic Proteins with Anciently Duplicated Paralogs

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate. Received: 30 December 1999 / Accepted: 5 April 2000     

On the Occurrence of Horizontal Gene Transfer Among an Arbitrarily Chosen Group of 26 Genes

The deduced amino acid sequences from 1200 Haemophilus influenzae genes was compared to a data set that contained the orfs from yeast, two different Archaea and the Gram+ and Gram− bacteria, Bacillus subtilis and Escherichia coli. The results of the comparison yielded a 26 orthologous gene set that had at least one representative from each of the four groups. A four taxa phylogenetic relationship for these 26 genes was determined. The statistical significance of each minimal tree was tested against the two alternative four taxa trees. The result was that four genes significantly supported the (Archaea, Eukaryota) (Gram+, Gram−) topology, two genes supported the one where Gram− and Eukaryota form a clade, and one gene supported the tree where Gram+ and Eukaryota define one clade. The remaining genes do not uniquely support any phylogeny, thereby collapsing the two central nodes into a single node. These are referred to as star phylogenies. I offer a new suggestion for the mechanism that gave rise to the star phylogenies. Namely, these are genes that are younger than the underlying lineages that currently harbor them. This hypothesis is examined with two proteins that display the star phylogeny; namely onithine transcarbamylase and tryptophan synthetase. It is shown, using the distance matrix rate test, that the rate of evolution of these two proteins is comparable to a control gene when rates are determined by comparing closely related species. This implies that the genes under comparison experience comparable functional constraint. However, when the genes from remotely related species are compared, a plateau is encountered. Since we see no unusual levels of functional constraint this plateau cannot be attributed to the divergence of the protein having reached saturation. The simplest explanation is that the genes displaying the star phylogenies were introduced after Archaea, Eukaryota, and Bacteria had diverged from one another. They presumably spread through life by horizontal gene transfer. Received: 12 July 2001 / Accepted: 27 July 2001     

Evolutionary Relationship Between Translation Initiation Factor eIF-2γ and Selenocysteine-Specific Elongation Factor SELB: Change of Function in Translation Factors

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the γ subunit of initiation factor 2 (eIF-2γ), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2γ gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2γ within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2γ proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2γ/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life. Received: 2 January 1998 / Accepted: 1 June 1998     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

The Archaea Monophyly Issue: A Phylogeny of Translational Elongation Factor G(2) Sequences Inferred from an Optimized Selection of Alignment Positions

A global alignment of EF-G(2) sequences was corrected by reference to protein structure. The selection of characters eligible for construction of phylogenetic trees was optimized by searching for regions arising from the artifactual matching of sequence segments unique to different phylogenetic domains. The spurious matchings were identified by comparing all sections of the global alignment with a comprehensive inventory of significant binary alignments obtained by BLAST probing of the DNA and protein databases with representative EF-G(2) sequences. In three discrete alignment blocks (one in domain II and two in domain IV), the alignment of the bacterial sequences with those of Archaea–Eucarya was not retrieved by database probing with EF-G(2) sequences, and no EF-G homologue of the EF-2 sequence segments was detected by using partial EF-G(2) sequences as probes in BLAST/FASTA searches. The two domain IV regions (one of which comprises the ADP-ribosylatable site of EF-2) are almost certainly due to the artifactual alignment of insertion segments that are unique to Bacteria and to Archaea–Eucarya. Phylogenetic trees have been constructed from the global alignment after deselecting positions encompassing the unretrieved, spuriously aligned regions, as well as positions arising from misalignment of the G′ and G″ subdomain insertion segments flanking the ``fifth' consensus motif of the G domain (?varsson, 1995). The results show inconsistencies between trees inferred by alternative methods and alternative (DNA and protein) data sets with regard to Archaea being a monophyletic or paraphyletic grouping. Both maximum-likelihood and maximum-parsimony methods do not allow discrimination (by log-likelihood difference and difference in number of inferred substitutions) between the conflicting (monophyletic vs. paraphyletic Archaea) topologies. No specific EF-2 insertions (or terminal accretions) supporting a crenarchaeal–eucaryal clade are detectable in the new EF-G(2) sequence alignment.     

Primary Structure and Phylogenetic Relationships of a Malate Dehydrogenase Gene from Giardia lamblia

The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria, archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within a eukaryote cytosolic MDH clade, to the exclusion of chloroplast, mitochondrial, and peroxisomal homologues. Furthermore, G. lamblia MDH is specifically related to a homologue from Trichomonas vaginalis. This MDH topology, together with published phylogenetic analyses of β-tubulin, chaperonin 60, valyl-tRNA synthetase, and EF-1α, suggests a sister-group relationship between diplomonads and parabasalids. Since these amitochondriate lineages contain genes encoding proteins which are characteristic of mitochondria and α-proteobacteria, their shared ancestry suggests that mitochondrial properties were lost in the common ancestor of both groups. Received: 14 September 1998 / Accepted: 29 December 1998     

The Molecular Evolutionary History of a Winged Bean α-Chymotrypsin Inhibitor and Modeling of Its Mutations Through Structural Analyses

A serine protease inhibitor of the Kunitz-STI (soybean trypsin inhibitor) family, isolated from the legume seeds of winged bean, was found to inhibit chymotrypsin at a 1:2 stoichiometric ratio. When the structure was determined in our laboratory, it was found to form a characteristic β-trefoil fold, which is also seen in other proteins from distant families and sources. The folding organization divides the protein into three approximately equal subdomains related by a pseudo-threefold axis of symmetry passing parallel to the barrel axis of the trefoil. Following the now established idea that the present-day genes originated from ancestral minigenes through evolution, the origin of the proteins having this β-trefoil organization is scrutinized using its subdomain motif as the search probe. The results, based mainly on structural analyses, indicate the independent existence of such a motif, mimicking the unknown ancestral protein(s) that might have been distributed in nature, not only by gene duplication, but also by insertion and permutation in other folds. The understanding led to a hypothesis for the possible origin of the Kunitz-STI family. On the basis of this model of evolution, structurally hypervariable regions were located on the protein where mutations could be designed and a broad range of engineering of the protein's activity could be conceived. Received: 20 January 1999 / Accepted: 6 October 1999