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The canonical network of autotrophic intermediary metabolism: minimal metabolome of a reductive chemoautotroph.

Introduction

Metabolic charts have been a central feature in the study of biochemistry since Donald Nicholson developed his first Metabolic Pathways Chart in 1955 (Nicholson, 2006). A printed version was published and circulated by the Koch-Light Laboratories starting in 1960. A little-recognized feature is that although there are tens of millions of taxa, for most purposes there is a single metabolic pathways chart. The subject was formalized even further when "An Introduction to Metabolic Pathways" by S. Dagley and Donald Nicholson appeared in 1970 (Dagley and Nicholson, 1970). In the "Contents" of that document, the following taxa are referred to: mice, microorganisms, bacteria, Pseudomonadaceae, Enterobacteriacea, Chlorobium thiosulfatophilum, Proprionibacterium shermanii, E. coli, yeast, and mammals. The formalism thus was developed for heterotrophic oxidizers. Even though not universal, the chart covers a broad range of organisms.

In the course of studying biogenesis, we became aware of bacteria that were reductive autotrophs and fixed carbon from carbon dioxide through the reductive tricarboxylic acid (rTCA) cycle (Fuchs, 1989). Since these organisms synthesized all organics from carbon dioxide, hydrogen, ammonia, and hydrogen sulfide, it seemed apparent that a chart of the metabolic pathways of these organisms would make an interesting comparison to oxidative heterotrophs and might point to the last universal common ancestor. The autotrophic bacterium Aquifex aeolicus had been completely sequenced, was partially annotated, lay near the root of the ribosomal phylogenetic tree (Deckert et al., 1998), and was available on the KEGG database (KEGG Pathway Database, 1995). We developed the pathways chart for this taxon, compared it with that for other sequenced reductive autotrophs, and developed a canonical chart for this class of organisms. This chart can then be examined from the point of view of biogenesis and also can be compared with the anabolic features of oxidative heterotrophs and photoautotrophs to look for robust universal features of metabolism that have persisted for several billion years. In this paper we present the chart developed from the KEGG database, and in a subsequent paper we will discuss the empirical generalizations that can be drawn from the information in the family of pathway charts (Srinivasan and Morowitz, unpubl. data).

Autotrophic carbon fixation and extremophily are recognized to be two likely hallmarks of primordial organisms (Hugler et al., 2003; Pakchung et al., 2006). Thus, the most favored candidates for investigating the origins and earliest evolution of life are the extremophiles, and in particular the hyperthermophilic organisms (Hugler et al., 2003). Reviewing the list of autotrophic extremophiles whose genomes have been completely sequenced and are available in the public domain, we selected Aquifex aeolicus as the central candidate for developing a complete chart of anabolic intermediary metabolism. Such a chart constitutes a representative minimal metabolome--a collection of all the small molecules expressed by an organism over time--of reductive chemoautotrophic bacteria. Our choice of Aquifex is further justified by its placement at the deepest phylogenetic branch point, closest to the last universal common ancestor (Burggraf et al., 1992), and by the fact that it is among the known thermophilic chemoautotrophs whose genomes have been completely sequenced,

A. aeolicus has one of the smallest genomes among free-living bacteria and the smallest among free-living autotrophic bacteria, indicating limited, if not minimal, metabolic flexibility. We compared its genome annotations and available biochemical information with those of other representative organisms related to chemoautotrophy (which include Thiomicrospira denitrificans, Hydrogenobacter thermophilus, and an archeaon--Pyrobaculum aerophilum), and extracted and integrated relevant information to formulate a canonical metabolic chart. As our focus is restricted to only the conserved core intermediary metabolic network, any noise imposed by horizontal gene transfer is essentially eliminated or expected to be negligible. In this report, we present a metabolic network that represents the chart of intermediary metabolism of reductive chemoautotrophic bacteria. Also presented is a compilation of biochemical reactions and a list of metabolites enumerating a representative minimal metabolome of a free-living reductive chemoautotroph.

The chemoautorophic organisms used in constructing the canonical metabolic chart obtain their energy from redox couples in the environment leading to generation of ATP, nicotinamide adenine dinucleotide (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH). By coupling the oxidation and reduction of inorganic compounds to the generation of biomass, the activities of these organisms tie the geochemical transformation of their redox substances to the cycles of carbon, nitrogen, and phosphorus. Since our interest is in the core metabolic chart for anabolism, we will not include the energy pathways, but will assume the presence of redox couples that make ATP, NADH, and NADPH. Thus, we are focusing on the informatic as contrasted to the energetic aspects of metabolism.

Materials and Methods

All information regarding the pathways, the enzymes, the reactants, and the products for all the organisms whose genomes have been completely sequenced was obtained by data-mining the Kyoto Encyclopedia of Genes and Genomes (KEGG) database--an online, public domain, open-source collection of databases that includes metabolic pathways, genome sequences, and analysis (Kanehisa, 1997; Kanehisa and Goto, 2000). The KEGG Pathway Database (1995) was utilized for retrieval of all information, using the search criteria related to "organisms" "gene catalogs," and "KEGG Orthology" under "network hierarchy" and "metabolism," and the entries for each of the enzymes mentioned therein. Numbers in parentheses after enzyme names are assigned by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology on the basis of the reactions that they catalyze.

We have extracted pathway information for the anabolic synthesis of the fundamental types of monomers: amino acids, sugars, nucleotide derivatives, and lipids from the KEGG Pathway database. For all the relevant organisms examined, a comparative analysis revealed a consensus in the linear progression of compounds by stepwise synthesis for each type of monomer; this consensus enabled us to construct a minimal metabolic chart for an autotroph (see Supplemental Figure 1 at http://www.biolbull.org/supplemental/). The genome annotation of every organism sequenced is continually being updated as new information becomes available from biochemical investigations and more sequence data, either on the organism itself or from related organisms and allied studies. In the KEGG biosynthetic pathway maps, we have observed a few monomers wherein the enzyme annotations have gaps, implying lack of a positive identification for a specific enzyme or enzymes that make a particular intermediate. However, both the enzyme that is required in the previous step to synthesize the precursor compound and the enzyme that subsequently processes the aforementioned specific intermediate to the next step are present and characterized. Let us consider a pathway in which initial compound A is sequentially progressed to end product Z. Along the way, compound P is converted to Q, which is processed to R, and so on in the cascade. In this example, the enzyme, that converts P to Q may be uncharacterized, although the enzymes required to make P and the one that processes Q to R have been characterized and annotated. In such instances, the comparative pathway analysis utilizing data from annotation of other taxa enables completion of the pathway map by inference. We have adopted this strategy in the comparative analysis of metabolic pathways for the above-mentioned organisms in order to construct the canonical metabolic chart of a reductive chemoautotroph.

[FIGURE 1 OMITTED]

Results and Discussion

Chemolithoautotrophs are organisms that acquire energy by redox reactions of inorganic compounds and utilize O[C.sub.2] as the sole source of carbon for anabolic synthesis of compounds of intermediary metabolism for growth. At the present time, at least five autotrophic mechanisms have been well established for carbon assimilation (Thauer, 2007; Berg et al., 2007); among these, the reductive TCA cycle (rTCA) is the pathway utilized by many reductive chemoautotrophs (Fuchs, 1989; Nakagawa and Takai, 2008). Recently, in Aquificales, which is a strictly thermophilic bacterial lineage of chemoautotrophs discovered at hydrothermal vents worldwide, the rTCA-based mechanism of carbon fixation has been identified in all three families of the phylum (Hugler et al., 2007). This lineage that is placed phylogentically near the root includes Aquifex aeolicus, Thiomicrospira denitrificans, and Hydrogenobacter thermophilus, the main reference organisms that contributed to the construction of the complete anabolic chart of metabolism. Figure 1 depicts the reductive TCA cycle, which may be considered the core of intermediary metabolism (Fig. 1 is taken from the complete anabolic chart of intermediary metabolism in Supplemental Fig. 1, provided online at http://www.biolbull.org/supplemental/). The rTCA cycle is network-autocatalytic and can self-amplify, promoting growth (Smith and Morowitz, 2004). A supply of one equivalent of any of the main components of this cycle yields two equivalents of the same component at the end of the cycle.

Table 1 details the 12 biochemical steps of the rTCA cycle, which are a subset of a defined class of organic chemical reactions (see Fig. 1). It is instructive to note that the biochemistry of intermediary metabolism constitutes a relatively sparse set of chemical reactions. All these reactions are catalyzed by enzymes. Four of the 12 steps are the relatively simple addition or removal of water, while the other 8 are mediated by specific cofactors. Cofactors are a special class of molecules that we term chimeromers (see below), which function as construction enablers in synthesizing the components of the metabolome.
Table 1

Reactions of the reductive tricarboxylic acid (rTCA) cycle

Reaction  Reaction                Compounds      Compound  Compound
number                                            class *   number

1         Citrate + CoA + ATP    Acetyl-CoA         NC        1
          [??] Acetyl-CoA +
          Oxaloacetate +
          Orthophosphate
                                 Oxaloacetate       NC        2

2         2 Ferredoxin           Pyruvate           NC        3
          (reduced) +
          Acetyl-CoA +
          [CO.sub.2] + 2
          [H.sup.+] [??]
          2 Ferredoxin
          (oxidized)
          + Pyruvate + CoA

3         Pyruvate + ATP +       Phosphoenol        NC        4
          [H.sub.2]O [??]        pyruvate
          Phosphoenolopyruvate
          + AMP +
          Orthophosphate

4         [H.sub.2]O +           Oxaloacetate       NC        2
          Phosphoenolpyruvate +
          [CO.sub.2] [??]
          Orthophosphate +
          Oxaloacetate

5         Oxaloacetate + NADH +  Malate             C         5
          [H.sup.+] [??]
          Malate +
          [NAD.sup.+]

6         Malate [??]            Fumarate           NC        6
          Fumarate
          + [H.sub.2]O

7         FADH2 + Fumarate       Succinate          C         7
          [??] Succinate +
          FAD

8         ATP + Succinate + CoA  Succinyl-CoA       C         8
          [??]
          ADP +
          Orthophosphate +
          Succinyl-CoA

9         Ferredoxin (reduced)   2-Oxoglutarate     NC        9
          + Succinyl-CoA +
          [CO.sub.2] [??]
          Ferredoxin (oxidized)
          + 2-Oxoglutarate +
          CoA

10        2-Oxoglutarate +       Isocitrate         C         10
          NADPH + [H.sup.+]
          [??]
          Isocitrate +
          [NADP.sup.+]

11        Isocitrate [??]        cis-Aconitate      C         11
          cis-Aconitate +
          [H.sub.2]O

12        cis-Aconitate +        Citrate            C         12
          [H.sub.2]O [??]
          Citrate

* C, Core; NC, Nodel Core.


The rTCA cycle shown in Figure 1 is the core of synthesis in autotrophic organisms and supplies source compounds for the building blocks that undergo condensation and polymerization reactions to yield macromolecules. This autocatalytic cycle has five major source nodes (i) pyruvate, (ii) phosphoenolpyruvate, (iii) acetyl-CoA, (iv) 2-oxoglutarate, and (v) oxaloacetate. All anabolic pathways have their origins in these five source nodes for the synthesis of the entire metabolome.

The components of a chemoautotrophic metabolome can be grouped as follows. The nodal molecules of the rTCA cycle are the starting compounds and go through a progressive process via specific biosynthetic pathways in which they are modified stepwise as chemical intermediates in a limited set of chemical reactions (see Fig. l and Supplemental Fig. 1)-- eventually yielding monomers, which are small molecules ([approximately equal to]50-500 daltons). Most of the monomers serve as building blocks that undergo iterative dehydration reactions to generate mid-sized as well as long-chain polymers (several kilodaltons) that are proteins, DNA, RNA, carbohydrates, isoprenoids, and peptidoglycan (which makes up the cell wall). Under the functional classification presented in Supplemental Table 1 (http://www.biolbull.org/supplemental/), most basic building-block monomers, for example Leu (leucine) and dATP (2'-deoxyadenosine 5'-triphosphate), belong to the class of "Precursors to Polymerization" (PP); these compounds do not undergo any further chemical transformation other than polymerization. Other monomers, such as aspartic acid and guanosine triphosphate (GTP), are termed "Nodal Precursors to Polymerization" (NPP), and as the name implies, these compounds are utilized in more than one reaction in addition to polymerization. That is, they may be further modified and integrated, totally or partially, with other compounds--for example, in condensation reactions. The rest of the metabolome components are pathway intermediates (I) such as 2-isopropyl malate and orotate, most of which do not undergo more than one chemical transformation; the subset of compounds that are nodal in this class of intermediates are termed "Nodal Intermediates "(NT), for example, phosphoenol pyruvate and 5-phosphoribosyl-l-pyrophosphate (PRPP). These compounds are generated in one chemical reaction and processed and modified in more than one reaction. The lipid constituents of the cell membrane are divided into "Lipid Intermediates" (LI) and "Lipid Components" (LC), which are the terminal lipid monomers.

A small subset of monomers, conventionally known as cofactors, are a part of and essential to the making of all metabolomes. These are terminal monomers and belong to a distinct class of compounds that we propose to call chimeromers. In any given metabolome, most of the cofactors are heterocyclic compounds, and in particular in the autotrophic metabolome under discussion, all of them are heterocyclic nitrogenous compounds. Unlike the monomers of the three major building-block categories that are synthesized by compound progression of rTCA cycle nodal molecules, the cofactors are synthesized starting from monomers and intermediates. These are transformed to yield heterocyclic compounds that are analogs of the ring systems of pyridine, pyrimidine, thiazole, pterin, and isoalloxazine (Begley, 2006). These compounds appear to be a noncongruent mosaic of members of monomers--namely amino acids, nucleotides, and their pathway intermediates. Typical examples are CoA, that has an AMP handle bridged through pantothenic acid to a modified (decarboxylated) cysteine and S-adenosyl-L-methionine (SAM) which is a direct chimera of ATP and methionine. In addition to CoA and SAM, many other cofactors like NAD, NADP, FAD, and ATP are all nucleotides or contain heterocyclic nitrogenous bases as seen in thiamin diphosphate (ThPP) and tetrahydrofolate (THF). While the intermediates of chimeromer synthesis are abbreviated as I, the terminal chimeromers that are the cofactors are denoted by CF in Supplemental Table 1 (online at http://www.biolbull.org/supplemental/). We have introduced the term chimeromers because there does not seem to be an existing term that describes molecules made of mixed monomers of different classes.

Table 2 details the distribution of the compounds of an autrotrophic metabolome that totals to 287. More than half of the compounds are intermediates in the biosynthesis of the monomelic building blocks that polymerize to yield macromolecules. A network diagram of the anabolic synthesis of all these compounds and their pathways are presented in Supplemental Figure I (online at http://www.biolbull.org/supplemental/).The reactions from which this network was constructed are presented in Supplemental Table 1 (at http://www.biolbull.org/supplemental/). This network portrays the canonical chart of autotrophic intermediary metabolism that composes the minimal metabolome of a reductive chemoautotroph. Among the different types of chemical reactions required, a sparse set of simple reactions that dominate and cause chemical transformations stepwise and advance the start compounds through intermediates to the final monomers are represented by arrows that are color-coded (see Supplemental Figure 1, at http://www.biolbull.org/supplemental/). These reactions include (i) oxidation-reduction, (ii) carboxylation-decarboxylation, (iii) hydrolysis-dehydration, (iv) phosphorylation-dephosphorylation, (v) amination, and (vi) acylation; all these reactions are enabled by specific cofactors in enzyme-catalyzed transformations. While we attempt to delineate principles that may govern the selection of these compounds and the evolution of the autotrophic network, simple analysis has yielded several empirical correlations that would aid in deriving the set of selection rules. These features will be detailed in a follow-up publication (Morowitz and Srinivasan, unpubl. data).
Table 2
Distribution of metabolome compounds

Compound type                      Number of compounds

Core                                        6
Nodel Core                                  7
Intermediate                              169
Nodal Intermediate                         20
Precursor to Polymerization                18
Nodel Precursor to Polymerization          13
CoFactor                                   12
Lipid Intermediate                         35
Lipid Component                             7

Total                                     287


Acknowledgments

We are indebted to the National Science Foundation (FIBR Grant) and William Melton for their support.

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Vijayasarathy Srinivasan * AND Harold J. Morowitz

Krasnow Institute for Advanced Studies, George Mason University, Fairfax, Virginia 22030

* To whom correspondence should be addressed: E-mail: vsriniva@gmu.edu

Received 12 September 2008; accepted 26 November 2008.
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Author:Srinivasan, Vijayasarathy; Morowitz, Harold J.
Publication:The Biological Bulletin
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Date:Apr 1, 2009
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