Phylogenetic Analysis of Metabolic Pathways

The information provided by completely sequenced genomes can yield insights into the multi-level organization of organisms and their evolution. At the lowest level of molecular organization individual enzymes are formed, often through assembly of multiple polypeptides. At a higher level, sets of enzymes group into metabolic networks. Much has been learned about the relationship of species from phylogenetic trees comparing individual enzymes. In this article we extend conventional phylogenetic analysis of individual enzymes in different organisms to the organisms' metabolic networks. For this purpose we suggest a method that combines sequence information with information about the underlying reaction networks. A distance between pathways is defined as incorporating distances between substrates and distances between corresponding enzymes. The new analysis is applied to electron-transfer and amino acid biosynthesis networks yielding a more comprehensive understanding of similarities and differences between organisms. Received: 14 August 2000 / Accepted: 4 January 2001     

Deriving phylogenetic trees from the similarity analysis of metabolic pathways

MOTIVATION: Comparative analysis of metabolic pathways in different genomes can give insights into the understanding of evolutionary and organizational relationships among species. This type of analysis allows one to measure the evolution of complete processes (with different functional roles) rather than the individual elements of a conventional analysis. We present a new technique for the phylogenetic analysis of metabolic pathways based on the topology of the underlying graphs. A distance measure between graphs is defined using the similarity between nodes of the graphs and the structural relationship between them. This distance measure is applied to the enzyme-enzyme relational graphs derived from metabolic pathways. Using this approach, pathways and group of pathways of different organisms are compared to each other and the resulting distance matrix is used to obtain a phylogenetic tree. RESULTS: We apply the method to the Citric Acid Cycle and the Glycolysis pathways of different groups of organisms, as well as to the Carbohydrate metabolic networks. Phylogenetic trees obtained from the experiments were close to existing phylogenies and revealed interesting relationships among organisms.     

Construction of phylogenetic trees by kernel-based comparative analysis of metabolic networks

Background  

To infer the tree of life requires knowledge of the common characteristics of each species descended from a common ancestor as the measuring criteria and a method to calculate the distance between the resulting values of each measure. Conventional phylogenetic analysis based on genomic sequences provides information about the genetic relationships between different organisms. In contrast, comparative analysis of metabolic pathways in different organisms can yield insights into their functional relationships under different physiological conditions. However, evaluating the similarities or differences between metabolic networks is a computationally challenging problem, and systematic methods of doing this are desirable. Here we introduce a graph-kernel method for computing the similarity between metabolic networks in polynomial time, and use it to profile metabolic pathways and to construct phylogenetic trees.     

MRAD: Metabolic reaction analysis database--an entity-relationship approach

The Metabolic Reaction Analysis Database (MRAD) is a relational database based on the Entity-Relationship (ER) model which combines information about organisms, biochemical pathways, reactions, enzymes, substrates, products and genes. It describes 244,596 genes in 79 organisms, 6,552 enzymes, and 3,552 reactions, 3,100 substrates, 2,866 products and 118 metabolic pathways. The MRAD graphical user interface allows for the identification of metabolic reactions which are similar and dissimilar in multiple organisms, reactions in a pathway which are missing in an organism and using any combination between one to six of the biological entities of organisms, genes, pathways, enzymes, substrates and products to determine metabolic reactions. MRAD provides a powerful and efficient tool for the construction of flux balance models for metabolic engineering applications.     

Detecting network communities: an application to phylogenetic analysis

This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis.     

The Origin and Evolution of Metabolic Pathways: Why and How did Primordial Cells Construct Metabolic Routes?

The emergence and evolution of metabolic pathways represented a crucial step in molecular and cellular evolution. In fact, the exhaustion of the prebiotic supply of amino acids and other compounds that were likely present on the primordial Earth imposed an important selective pressure, favoring those primordial heterotrophic cells that became able to synthesize those molecules. Thus, the emergence of metabolic pathways allowed primitive organisms to become increasingly less dependent on exogenous sources of organic compounds. Comparative analyses of genes and genomes from organisms belonging to Archaea, Bacteria, and Eukarya reveal that, during evolution, different forces and molecular mechanisms might have driven the shaping of genomes and the emergence of new metabolic abilities. Among these gene elongations, gene and operon duplications played a crucial role since they can lead to the (immediate) appearance of new genetic material that, in turn, might undergo evolutionary divergence, giving rise to new genes coding for new metabolic abilities. Concerning the mechanisms of pathway assembly, both the analysis of completely sequenced genomes and directed evolution experiments strongly support the patchwork hypothesis, according to which metabolic pathways have been assembled through the recruitment of primitive enzymes that could react with a wide range of chemically related substrates. However, the analysis of the structure and organization of genes belonging to ancient metabolic pathways, such as histidine biosynthesis, suggests that other different hypothesis, i.e., the retrograde hypothesis, may account for the evolution of some steps within metabolic pathways.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree

We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.     

Functional Phylogeny: the Use of the Sensitivity of Ribosomes to Protein Synthesis Inhibitors as a Tool to Study the Evolution of Organisms

In order to study the functional phylogeny of organisms, forty different protein synthesis inhibitors with diverse domain and funcional specificities have been used to analyze forty archaeal, bacterial and eukaryotic translational systems. The inhibition curves generated with the different ribosome-antibiotic pairs have shown very interesting similarities among organisms belonging to the same phylogenetic group, confirming the feasibility of using such information in the development of evolutionary studies. A new method to extract most of the information contained in the inhibition curves is presented. Using a statistical treatment based on the principal components analysis of the data, we have defined coordinates for the organisms which have allowed us to perform a functional clustering of them. The phenograms obtained are very similar to those generated by 16/18S rRNA sequence comparison. These results prove the phylogenetic value of our functional analysis and suggest an interesting intersection between genotypic and phenotypic (functional) information.     

Characterization of two new facultative methanotrophs

Two new facultative methane-oxidizing bacteria have been isolated from lake water enrichments. The organisms have been characterized in terms of colony types, growth characteristics, the guanine plus cytosine content of their deoxyribonucleic acid, thin sections, oxidation rates, and carbon assimilation pathways. Methane-grown cells of both organisms contained intracytoplasmic membranes similar to those described as type II in other methanotrophic bacteria. Neither organism had such membranes when grown heterotrophically. Both organisms assimilated methane by way of the isocitrate lyase-negative serine pathway for formaldehyde incorporation. The enzymes of this pathway were high in specific activity in cells grown on methane and were at low levels in cells grown either on heterotrophic substrates or on heterotrophic substrates plus methane. It is proposed that both organisms be classified in the genus Methylobacterium as two new species, Methylobacterium ethanolicum and Methylobacterium hypolimneticum.     

Trypanosoma seryl-tRNA synthetase is a metazoan-like enzyme with high affinity for tRNASec

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma. First, the biological function of a putative Trypanosoma seryl-tRNA synthetase was characterized in vivo. Secondly, the molecular recognition by Trypanosoma seryl-tRNA synthetase of its cognate tRNAs was dissected in vitro. The cellular distribution of tRNA(Sec) was studied, and the catalytic constants of its aminoacylation were determined. These were found to be markedly different from those reported in other organisms, indicating that this reaction is particularly efficient in trypanosomatids. Our functional data were analyzed in the context of a new phylogenetic analysis of eukaryotic seryl-tRNA synthetases that includes Trypanosoma and Leishmania sequences. Our results show that trypanosomatid seryl-tRNA synthetases are functionally and evolutionarily more closely related to their metazoan homologous enzymes than to other eukaryotic enzymes. This conclusion is supported by sequence synapomorphies that clearly connect metazoan and trypanosomatid seryl-tRNA synthetases.     

Biological diversity,chemical mechanisms,and the evolutionary origins of bioluminescent systems

A diversity of organisms are endowed with the ability to emit light, and to display and control it in a variety of ways. Most of the luciferins (substrates) of the various phylogenetically distant systems fall into unrelated chemical classes, and, based on still limited data, the luciferases (enzymes) and reaction mechanisms are distinctly different. Based on its diversity and phylogenetic distribution, it is estimated that bioluminescence may have arisen independently as many as 30 times in the course of evolution. However, there are several examples of cross-phyletic similarities among the substrates; some of these may be accounted for nutritionally, but in other cases they may have evolved independently.     

Evolutionary analysis of fructose 2,6-bisphosphate metabolism

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.     

Microbial consortia involved in the anaerobicdegradation of hydrocarbons

In this review, we examine the energetics of well-characterized biodegradation pathways and explore the possibilities for these to support growth of multiple organisms interacting in consortia. The relevant phenotypic and/or phylogenetic characteristics of isolates and consortia mediating hydrocarbon degradation coupled with different terminal electron-acceptingprocesses (TEAP) are also reviewed. While the information on metabolic pathways has been gained from the analysis of individual isolates, the energetic framework presented here demonstrates that microbial consortia could be readily postulated for hydrocarbon degradation coupled to any TEAP. Several specialized reactions occur within these pathways, and the organisms mediating these are likely to play a key role in defining the hydrocarbon degradation characteristics of the community under a given TEAP. Comparing these processes within and between TEAPs reveals biological unity in that divergent phylotypes display similar degradation mechanisms and biological diversity in that hydrocarbon-degraders closely related as phylotypes differ in the type and variety of hydrocarbon degradation pathways they possess. Analysis of microcosms and of field samples suggests that we have only begun to reveal the diversity of organisms mediating anaerobic hydrocarbon degradation. Advancements in the understanding of how hydrocarbon-degrading communities function will be significantly affected by the extent to which organisms mediating specialized reactions can be identified, and tools developed to allow their study in situ.     

The evolution of function: a new method to assess the phylogenetic value of ribosomal sensitivity to antibiotics.

Thirty-five archaeal, bacterial and eukaryotic translational systems have been proved against forty different protein synthesis inhibitors with diverse domain and functional specificities. The inhibition curves generated in every ribosome-antibiotic combination had previously shown interesting similarities among organisms belonging to the same phylogenetic group. This opened the possibility of using such functional information for developing evolutionary studies. A new mathematical method based on the main data components analysis has been developed to extract most of the information contained in the inhibition curves. The phenograms obtained closely resemble those generated by the small ribosomal subunit rRNA sequence comparison and such functional clustering is also congruent when a particular subset of organisms and/or antibiotics is used. These results prove the phylogenetic value of our functional analysis and suggest that the ribosome represents an interesting intersection between genotypic and phenotypic (functional) information stored in organisms.     

Phylogenetic comparison of metabolic capacities of organisms at genome level

Horizontal gene transfer (HGT) has been shown to widely spread in organisms by comparative genomic studies. However, its effect on the phylogenetic relationship of organisms, especially at a system level of different cellular functions, is still not well understood. In this work, we have constructed phylogenetic trees based on the enzyme, reaction, and gene contents of metabolic networks reconstructed from annotated genome information of 82 sequenced organisms. Results from different phylogenetic distance definitions and based on three different functional subsystems (i.e., metabolism, cellular processes, information storage and processing) were compared. Results based on the three different functional subsystems give different pictures on the phylogenetic relationship of organisms, reflecting the different extents of HGT in the different functional systems. In general, horizontal transfer is prevailing in genes for metabolism, but less in genes for information processing. Nevertheless, the major results of metabolic network-based phylogenetic trees are in good agreement with the tree based on 16S rRNA and genome trees, confirming the three domain classification and the close relationship between eukaryotes and archaea at the level of metabolic networks. These results strongly support the hypothesis that although HGT is widely distributed, it is nevertheless constrained by certain pre-existing metabolic organization principle(s) during the evolution. Further research is needed to identify the organization principle and constraints of metabolic network on HGT which have large impacts on understanding the evolution of life and in purposefully manipulating cellular metabolism.     

Identification of a hydratase and a class II aldolase involved in biodegradation of the organic solvent tetralin

Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated thnE and thnF, are located downstream of the previously identified thnD gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.