Branched chain amino acid biosynthesis.

<p>The chemical sequences show the parallels in terms of local functional group chemistry within the reconstructed ancestral pathways to valine, leucine and isoleucine. The blue box highlight the citramalate pathway of -ketobutyrate synthesis, reconstructed here to represent the ancestral sequence to this compound. The molecules highlighted in orange and green in turn show the compact interconnectedness of the ancestral pathways to the branched chain amino acids. Parallels to substrate sequences within the oxidative TCA are also highlighted, as well as the alternate route to -ketobutyrate from threonine.</p

Summary of metabolism.

<p>Reactions (highlighted in blue) represent, as we show here, the ancestral route to glycine and serine. In organisms that use the Wood-Ljungdahl pathway as the exclusive route to carbon-fixation, the direct reduction of also culminates in the synthesis of acetyl-CoA, a step absent in many other organisms that do employ the preceding parts of the pathway. Reactions constitute the oxidative route to serine and glycine, which are subsequently cleaved to supply metabolism in many late-branching bacteria and eukaryotes. Reaction constitutes the glyoxylate pathways, important in cyanobacteria and photorespiring plants. Abbreviations: MFR, methanofuran; , tetrahydromethanopterin; THF, tetrahydrofolate; MET, methionine; LA, lipoic acid; GLY, glycine; SER, serine; PSR, phosphoserine; PHP, 3-phosphohydroxypyruvate; 3PG, 3-phosphoglycerate; GLX, glyoxylate. For a more detailed figure and caption, as well as names and EC classes of numbered reactions see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi.1002455.s001" target="_blank">Text S1</a> and Fig. S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi.1002455.s001" target="_blank">Text S1</a>.</p

Phylometabolic tree of carbon-fixation.

<p>The grey line represents the maximum-parsimony tree linking the major metabolic phenotypes (diagrams). Three major modules shown schematically (see the legend) are pterin/folate- metabolism, the pentose-phosphate pathway, and the TCA cycle reactions. Lost reactions (symbol) include the acetyl-CoA synthase (in metabolism), and ferredoxin-dependent succinyl-CoA synthase (in TCA loop) or citryl-CoA synthase (not shown). Abbreviations: , formyl; , methylene; ACA, acetyl-CoA; PYR, pyruvate; GAP, glyceraldehyde-3-phosphate; F6B, fructose-1,6-bisphosphate; RIB, ribose-phosphate; RBL, ribulose-phosphate; ALK, alkalinity; others as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi-1002455-g002" target="_blank">Fig. 2</a>. Arrows indicate reaction directions; dashed line connecting 3PG to SER indicates intermittent or bidirectional reaction. Highlighted in yellow are the innovations underlying divergences, while red labels on links indicate evolutionary force associated with each innovation, explained in the text. Beneath each diagram is an extant species or clade name where the phenotype is found. Colored regions indicate domains within which all known instances of the indicated phenotypes are restricted.</p

Principles of Phylometabolic Analysis.

<p>Panel A shows how phylogenetic distributions of pathways helps interpret the curation of individual metabolic networks. In this case comparison of metabolic gene profiles suggests the orange pathway represents the correct completion. Panel B in turn shows how pathway distributions in turn also suggest evolutionary sequences. Imposing continuity of metabolite production at the ecosystem level allows us to represent those sequences as phylometabolic trees, in which each node represents a functional phenotype with an explicit internal chemical structure.</p

Carbon-fixation in <i>Aquifex aeolicus</i>.

<p>The main fixation pathway is the reductive citric acid (rTCA) cycle, from which most anabolic pathways are initiated. Reductive folate chemistry is a secondary fixation pathway from which an additional small set of anabolic pathways is initiated (R = aminobenzoate-derived side chain). Whether formate attachment occurs at the or position of THF remains to be elucidated (see text). Relative to the reconstructed root of carbon-fixation, in which the rTCA cycle and Wood-Ljungdahl pathway are fully integrated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087950#pone.0087950-Braakman2" target="_blank">[2]</a>, this hybrid strategy employed by <i>A. aeolicus</i> lacks only the grey-dashed reaction (acetyl-CoA synthesis). Molecules highlighted in blue represent the “pillars of anabolism”, TCA intermediates from which the vast majority of anabolic pathways have been initiated throughout evolution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087950#pone.0087950-Smith1" target="_blank">[42]</a>. Highlighted in green is succinyl-CoA, which forms a precursor to pyrroles through a later derived pathway in some organisms (but not <i>A. aeolicus</i>). Highlighted in red are reaction sequences involving the same local functional group transformation that in <i>A. aeolicus</i> are catalyzed by closely related enzymes in both halves of the rTCA cycle. Green dashed arrows highlight alternate pathway sequences catalyzed by a single enzyme in other clades.</p

Distribution of rTCA enzymes in Aquificales.

<p>Numbers in each column represent the length of the enzyme in terms of amino acid residues, and the numbers in parentheses are the sequence similarities relative to the <i>T. thermophilus</i> version of those enzymes. Entries ** are for species that have only one copy of that enzyme class, which are aligned with the type to which it has greatest sequence similarity (sequence similarity for alternate alignment also shown for comparison). Families: I - Aquificaceae, II - Hydrogenothermaceae, III - Desulfurothermaceae. Abbreviations: ICDH, isocitrate dehydrogenase; BC, biotin carboxylase; -CoA synthase; LS, large subunit; SS, small subunit; PYR, pyruvate; AKG, -ketoglutarate; SUC-, succinyl-; CIT-, citryl.</p

Distribution of Isoleucine biosynthesis pathways.

<p>Distribution of Isoleucine biosynthesis pathways.</p

Phylogenetic distribution of direct pathways in Archaea and Bacteria.

<p>Dark purple dots indicate clades in which the complete gene complement for direct reductive synthesis of glycine and serine is found, light purple dots indicate clades where a complete pathway is suspected. Red dots indicate clades in which direct reduction of is known to be active, but in a form that lacks synthesis of glycine and serine. See main text for further details. The unrooted phylogenetic tree was adapted, with modification, from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi.1002455-Puigbo1" target="_blank">[14]</a>. In that work, the tree was created from an analysis of 102 ‘nearly universal’ clusters of orthologous groups of proteins (COGs) (present in of species in the tree) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi.1002455-Puigbo1" target="_blank">[14]</a>.</p

Principles of phylometabolic analysis.

<p><b>A</b> - <i>Example metabolic network structure</i>. Two essential metabolites, X and Y, can be synthesized through three known metabolic pathways, indicated by magenta, orange and purple arrows. Individual arrows represent enzymes catalyzing different steps in the pathway, and they can be either absent (empty arrows) or present (filled arrows) in the annotated genome of a particular strain R. Arrows in black are universal metabolic genes. <b>B</b> - <i>Phylogenetics constrains metabolic analysis</i>. Each branch in this phylogenetic tree represents an individual strain. The gap structure in the metabolic network of strain R, which was difficult to interpret in isolation becomes much less uncertain when placed in a phylogenetic context, suggesting that in this organism the orange pathway should be completed. <b>C</b> - <i>Metabolic analysis constrains phylogenetics</i>. Each branch in this tree represents a clade and individual (horizontal) rows within the metabolic gene profile matrices represent the profile of individual strains. Here the metabolic context suggests proper placement of a clade-level branch that was uncertain in an unconstrained phylogenetic tree. <b>D</b> - <i>Fully integrated phylometabolic tree</i>. Metabolic sequences are now drawn at the pathway level, and this tree represents a reconstruction of the evolution of the synthesis pathways of metabolites X and Y. Because mechanisms of regulation or heredity are not modeled, this phylometabolic tree is indifferent to distinctions of ecosystems or species, and the shown ‘phenotypes’ refer exclusively to the biochemistry of these pathways (see text for further details). <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002455#pcbi-1002455-g001" target="_blank">Fig. 1C</a> suggested that the orange pathway was ancestral, and that the magenta and purple pathways were derived from it. The only sequences that are then allowed under the constraint that essential metabolites X and Y are continuously produced, is the appearance of the purple and magenta pathways from stages in which they are co-present with the orange pathway.</p

Lipoic acid biosynthesis and lipoyl-protein assembly.

<p>In <i>E. coli</i> (green sequence), octanoate is transfered from ACP to the E2 subunit of pyruvate dehydrogenase (PDH) by LipB, followed by sulfuration to lipoic acid by LipA. In <i>E. coli</i> mutants lacking LipB, octanoate is transfered through an alternate route with an AMP-bound intermediate by LplA, normally used for incorporation of free lipoic acid. In <i>B. subtillis</i> (blue sequence), octanoate is transfered from ACP first to the H-protein of GCS by LipM, followed by a second transfer to the E2 subunit of PDH by LipL. <i>B. subtillis</i> also uses LplJ instead of LplA for incorporation of free lipoic acid. In red is the suggested ancestral biosynthesis of lipoic acid (see main text).</p