Literature Article Nature, 2010

Anonymous
timer Asked: Mar 5th, 2019
account_balance_wallet $9.99

Question Description

Explain the assigned literature article (Nature, 2010, 464, 1077), as if another student in CHEM 130B was your audience. An important component of scientific writing is to be concise, so you will have a 1-page limit to give a complete and accurate explanation. Remember that you may need to read sources that are cited or look at the Supporting Material for a full understanding of the paper. Please focus on the biochemical aspects of the paper rather than the phylogenetic studies, making sure to include key experiments and results.

Unformatted Attachment Preview

Vol 464 | 15 April 2010 | doi:10.1038/nature08884 LETTERS Fructose 1,6-bisphosphate aldolase/phosphatase may be an ancestral gluconeogenic enzyme Rafael F. Say1 & Georg Fuchs1 Most archaeal groups and deeply branching bacterial lineages harbour thermophilic organisms with a chemolithoautotrophic metabolism. They live at high temperatures in volcanic habitats at the expense of inorganic substances, often under anoxic conditions1. These autotrophic organisms use diverse carbon dioxide fixation mechanisms generating acetyl-coenzyme A, from which gluconeogenesis must start2–4. Here we show that virtually all archaeal groups as well as the deeply branching bacterial lineages contain a bifunctional fructose 1,6-bisphosphate (FBP) aldolase/phosphatase with both FBP aldolase and FBP phosphatase activity. This enzyme is missing in most other Bacteria and in Eukaryota, and is heat-stabile even in mesophilic marine Crenarchaeota. Its bifunctionality ensures that heat-labile triosephosphates are quickly removed and trapped in stabile fructose 6-phosphate, rendering gluconeogenesis unidirectional. We propose that this highly conserved, heat-stabile and bifunctional FBP aldolase/phosphatase represents the pacemaking ancestral gluconeogenic enzyme, and that in evolution gluconeogenesis preceded glycolysis5. The theory of a chemoautotrophic origin of life by transitionmetal-catalysed, autocatalytic carbon fixation assumes that chemoevolution took place in a hot volcanic flow setting6,7. It has become evident that in the phylogenetic tree of life the Archaea and deeply branching lineages of the Bacteria harbour thermophiles that thrive in volcanic environments on volcanic gases and inorganic substrates under anoxic or microaerobic conditions1 (Fig. 1). From carbon dioxide (CO2) or carbon monoxide (CO) they synthesize activated acetic acid, acetyl-coenzyme A (acetyl-CoA), as biosynthetic starting material. Their energy metabolism often makes use of hydrogen or CO as electron donors, and CO2 or sulphur compounds serve as electron acceptors in anaerobic respiration. Therefore, these chemolithoautotrophic organisms may serve as models for the study of primordial metabolism requiring the synthesis of organic building blocks from inorganic carbon. In none of these microorganisms does the Calvin–Benson–Bassham cycle seem to operate in CO2 fixation. Instead, other autotrophic pathways are functioning that have in common the formation of acetyl-CoA from inorganic carbon2–4. If so, the biosynthesis of sugars requires gluconeogenesis to start from acetyl-CoA as a precursor. Pyruvate and phosphoenolpyruvate (PEP) formation from acetyl-CoA and CO2 may differ2–4. In contrast, gluconeogenesis starting from PEP seems to be uniform. All enzyme activities and genes of a trunk Embden–Meyerhof–Parnas gluconeogenic pathway, which are necessary for the formation of FBP from PEP, are assumed to be present in Archaea. This is in contrast to the great diversity of glycolytic pathways and enzymes present in Archaea8. However, it has been generally difficult or impossible to detect FBP aldolase activity9. In many cases this enzyme activity could only be measured in the direction of FBP formation, whereas detection of the reverse reaction, FBP cleavage, has failed9,10. This discrepancy is puzzling as the FBP aldolase reaction is freely reversible. 1 Furthermore, tracer studies with several autotrophic Archaea revealed a labelling pattern of hexoses that was consistent with the classical gluconeogenic route involving FBP aldolase10,11. None of the archaeal genomes sequenced so far contains a classical FBP aldolase of class I (Schiff base intermediate, mainly found in Eukaryota) or class II (metal based catalysis, mainly in Bacteria and Fungi) (Supplementary Table 1). However, one small group harbours the gene encoding a different archaeal class IA aldolase12,13; this enzyme has a common evolutionary origin with class I and II aldolases14. Its function as FBP aldolase has been shown experimentally only in three cases generally by measuring the glycolytic direction. The other aldolase genes are only distantly related to this FBP aldolase and may have a different function (for example, in the archaeal aromatic biosynthesis pathway15; Supplementary Table 1). The majority of archaeal genomes sequenced so far lack any proven FBP aldolase gene, whereas generally the gene encoding an archaeal type V FBP phosphatase is present16 (Supplementary Table 1). This phosphatase also catalyses the sought-after FBP aldolase reaction, as the following experiments show. We searched for FBP aldolase plus FBP phosphatase activity in extracts from autotrophically grown cells of the thermophilic Archaea Ignicoccus hospitalis, Metallosphaera sedula and Thermoproteus neutrophilus, the central carbon metabolism of which has been studied recently2–4,10. The assay, at 65–85 uC, is based on the measurement of phosphate release, when triosephosphates were incubated with cell extracts. However, it was difficult to measure FBP aldolase activity owing to the heat instability of triosephosphates producing toxic methylglyoxal and forming phosphate, which interfered with the assay. The half-life of triosephosphates at 80 uC, pH 7, was confirmed to be 4 min12. Furthermore, we could not detect the reverse reaction; that is, FBP cleavage. However, FBP aldolase and phosphatase activities were readily detected using a discontinuous spectrophotometric assay for fructose 6-phosphate formation (for assay see Supplementary Information). The specific FBP aldolase activities were 0.02 mmol min21 (per mg of protein; 85 uC) in I. hospitalis, 0.006 mmol min21 (per mg of protein; 75 uC) in M. sedula, and 0.02 mmol min21 (per mg of protein; 85 uC) in T. neutrophilus. The specific FBP phosphatase activities were 1.5-fold as high. To convert efficiently the aldol reaction product FBP into fructose 6-phosphate, we added purified recombinant archaeal type V FBP phosphatase from I. hospitalis (Igni_0363) as auxiliary enzyme to the assay to pull forward the reaction. Only FBP phosphatase activity was observed when the intermediate FBP was supplied as substrate. Surprisingly, addition of triosephosphates resulted in a burst of FBP aldolase activity, even when cell extract was omitted. The archaeal type V FBP phosphatase has been studied in detail16,17 and its crystal structure was solved18; however, the main physiological function of the enzyme seems to have gone unnoticed. We meticulously purified the enzyme so that it showed no contaminating protein Mikrobiologie, Fakultät Biologie, Universität Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany. 1077 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS NATURE | Vol 464 | 15 April 2010 a Euryarchaeota Archaeoglobus fulgidus Thermoplasmatales * Halobacteriales Methanosarcinales Thermococcales Methanomicrobiales Methanopyrus kandleri * 68 Methanobacteriales 54 60 * Sulfolobales Ko Na no ar c eq hae ra u u cr rch ita m yp a ns to eu filu m m Methanococcales Desulfurococcales Thermoproteales * * * Mesophillic group 1 Crenarchaeota Crenarchaeota b Bacteria Cyanobacteria * Actinobacteria Firmicutes Fu so * ba ct ia 58 δ-Proteobacteria ε-Proteobacteria 29 62 Aquificae β-Proteobacteria 46 Thermotogae er hl C Bacteroidetes or ob i Deinococcus– Thermus Planctomycetes Spirochaetes Chlamydiae γ-Proteobacteria α-Proteobacteria Figure 1 | Phylogenetic unrooted trees of Archaea and Bacteria. Boxes indicate phyla containing FBP aldolase/phosphatase. Asterisks indicate phyla from which enzymes were studied. Only bootstrap values ,70% are indicated. a, Archaebacterial tree based on analyses of 64 conserved proteins (59 genomes). Red lines represent (hyper)thermophilic Archaea (.65 uC); blue lines mesophilic Archaea. Autotrophic lineages are marked with a dot. b, Eubacterial tree based on concatenated 37 ribosomal proteins (120 genomes30). Note that the systematic positions of the Thermotogae, Aquificae, Chloroflexi (not shown on the tree), the Deinococcus–Thermus lineage and in some respects also the deeply branching, thermophilic, acetogenic Clostridia/Firmicutes23 are considered as early branching22. (Supplementary Fig. 1 and Supplementary Table 2), but it still catalysed both aldolase and phosphatase reactions, demonstrating bifunctionality. The kinetic constants at 48 uC for I. hospitalis FBP aldolase activity were maximal velocity (vmax) of 0.54 mmol min21 (per mg of protein) and Michaelis constant (Km) of 0.23 mM for triosephosphates. The FBP phosphatase activity (vmax of 0.88 mmol min21 (per mg of protein); Km of 0.02 mM for FBP) was nearly twice as high as the aldolase activity, as observed in cell extract (for optimal pH see Supplementary Fig. 2). As further proof of bifunctionality, recombinant putative FBP phosphatases were overproduced and partly purified from five other Archaea covering different lineages (Fig. 2). We also expressed the synthetic FBP phosphatase gene of Cenarchaeum symbiosum19, a member of the marine group I Crenarchaeota (Fig. 2); similar genes allocated to this marine group occur in high numbers in the GOS databases. All six recombinant archaeal enzymes exhibited both FBP aldolase and phosphatase activities. This enzyme, now referred to as FBP aldolase/phosphatase, was previously considered as a potential determinant of hyperthermophily, besides reverse gyrase, a type I DNA topoisomerase able to stabilize DNA at high temperature by introducing positive supercoils20. Notably, even the Cenarchaeum enzyme, adapted to ocean temperature, was heat-stabile at 70 uC, with a half-life of 20 min at 82 uC, and the Ignicoccus enzyme even survived boiling for 1 h (Supplementary Fig. 3). The catalytic properties of the Cenarchaeum enzyme were analysed at 40 uC and are shown in Fig. 3. AMP, ADP or glucose (2 mM), known allosteric regulators of FBP phosphatases, had no effect. The lower activation energy of the Cenarchaeum enzyme compared to the Ignicoccus enzyme (Supplementary Fig. 4) indicates that these enzymes are adapted to the respective cold or hot optimal temperature for growth. The specific activity of the enzyme in cell extract is generally low, reflecting the minor need for carbohydrates in Archaea. Even in Escherichia coli, with its high content of sugars in lipopolysaccharides, the biosynthetic fluxes leading away from hexosephosphates add up to only ,13% of all biosynthetic fluxes. A data base search of sequenced genomes revealed that almost all Archaea contain the corresponding FBP aldolase/phosphatase gene (e-values ,10275), except for halophilic and a very few methanogenic Archaea that harbour, in most cases, the genes for other types of FBP aldolases and phosphatases (Supplementary Table 1). In most Archaea that do not grow on sugars the FBP aldolase/phosphatase gene is the only candidate gene for both FBP aldolase and FBP phosphatase. A look at the regulation of the FBP aldolase/phosphatase gene corroborates our expectation. Thermococcus kodakarensis forms this enzyme solely under gluconeogenic, but not under glycolytic, conditions16,17 (using the Embden–Meyerhof–Parnas pathway)8. A deletion mutant could grow under glycolytic, but not under gluconeogenic, conditions. Yet, complementation of this mutant by a different monofunctional FBP phosphatase did not restore growth under gluconeogenic conditions17. This is consistent with the notion that the deleted enzyme has, in addition, FBP aldolase activity. In contrast, Sulfolobus solfataricus uses a branched Entner–Doudoroff pathway for glycolysis8, in which FBP is not an intermediate, and therefore may tolerate constitutive expression of FBP aldolase/phosphatase21. A gene highly similar to the archaeal FBP aldolase/phosphatase gene (e-values ,10280) is also present in members of the deeply branching bacterial phyla22 (Fig. 2). They include genera of Aquificae (Aquifex, Hydrogenobaculum, Hydrogenivirga), Thermotogae (Petrotoga), Chloroflexi (Roseiflexus, Dehalococcoides), the Deinococcus–Thermus group (Thermus), as well as the mostly homoacetogenic, thermophilic Clostridia/Firmicutes (Carboxydibrachium, Thermoanaerobacter, Moorella, Pelotomaculum, Carboxydothermus, Natranaerobius) that also exhibit traits of a deeply branching phylum23,24 (for phylogenetic positions of the phyla see Fig. 1b). As a proof of concept, we overproduced the enzymes from Thermus thermophilus and Moorella thermoacetica and showed that they were an FBP aldolase/phosphatase, like the archaeal enzyme. There are only rare exceptions to the rule that this bifunctional enzyme is restricted to the Archaea and deeply branching, mostly thermophilic and autotrophic Bacteria (e-value ,10260). Probably all those bacteria have to perform a unidirectional gluconeogenesis from C2 or C3 compounds under some conditions, which may have favoured the acquisition of the gene by lateral transfer (for example, from mesophilic group 1 Crenarchaeota). Examples are the pathogenic Coxiella burnetii (c-Proteobacteria), the denitrifying Nitrococcus mobilis (c-Proteobacteria) and the symbiotic Bradyrhizobium japonicum (a-Proteobacteria) (Fig. 2). Syntrophs like Syntrophus aciditrophicus (d-Proteobacteria) depend on a close spatial contact with other Bacteria or (methanogenic) Archaea, which favours not only interspecies hydrogen transfer but also lateral gene transfer. Saccharopolyspora erythraea (Actinobacteria) contains many 1078 ©2010 Macmillan Publishers Limited. All rights reserved LETTERS mu um xie ja po lla ni c b he rm urn um e o aq phil tii u u ati s the cu Nitr rm s op oco h ccu Syntr s m ilus ophu obil s ac is iditro Hydro phic genob us aculu m sp. Aquifex ae olicus Hydrogenivirga sp. Na tra Th nae Aquificae st erm rob ius us ha r cc ‘M tus ida nd Ca is lud nnie lii ripa ma Sa us i ex M Methanopyrus kandleri Me tha no cal dococcus jan naschii Metha no co ccus ae Meth olicus ano c o Meth c cus v ano oltae coc Me cus tha va n oco cc ofl lor Ch De ha lo op co oly cc sp eth o or Me an a e ide tha or ryt s s eg no p hr ula cu ae . lleu a s m boo aris nei nig ’ ri Carboxydothermus hydrogenoformans m opropionicu ulum therm Pelotomac xviator’ dis auda ru a fo c ul ti oace tus ‘Des us therm Candida olic rella Moo than nsis e d u ge m pse con cter g cu a n b te cifi sp. ero ter pa ana m us lzii bac rmo ex hiu The ero o fl a c i n a a nh nes se ibr rmo Ro ste ge xyd a The o c no rb s a e u C h x et ifle es se id Ro co c co lo ha De eth an o gen m ka s s cu en oc nd sis c e en ro m p ng u i lf u uil su ofil aq ax e n m m D er e ga st um eu Th ldivir rot ndic isla Ca rmop ilus oph lum e u h u c e tr T oba us n Pyr rote s p ti o n o rm alidif The lum c bacu Pyro rophilum culum ae ba ro Py arsenaticum Pyrobaculum Thermoplasma volcanium 1 Thermoplasma volc anium 2 Thermop lasma ac idophilu Picro m philu s torr Meth idus anos Arc aeta hae therm oglo Pe oph tro bus ila tog Ac fulg am idu ob Ac iduli s il p is rof Th idu u l er nd m ipro um fu oc bo oc ndu on cu m ei sg b 1 am oon ei m 2 at ol er an s s . eu in sp s i ur s ns nn cu are oc us o ak c oc od us m coc us k hil er c o rop Th m ba oc er oc us us cc Th erm ios co fur Th rmo us e i occ Th yss roc s ab Py shii ccu oco oriko Pyr us h r sp. cocc obacte Pyro rm nothe Metha dmanae phaera sta Methanos r smithii Methanobrevibacte icutes Firm bi Co er is ns ke at ch ta Th zo rc ha eo Bac ter ia hi ur ya yr Candidatus ‘Korarchaeum cryptofilum’ Sulfolobus tokodaii Sulfolo bus ac idocald Meta llosp arius haera Sulf sedu olob la Su u s is lfolo la n Ign dicu bu s i s solf Hy cocc ata us ricu Ae pert ho s sp St rop herm ita y ap lis us hy rum bu lo pe tyl th icu rn er i x s m us m ar in us De in Th o c o er cc m u us s ad ta Br Cr en ar o ae ch um bios e sym ot aeum hae arc arch us n Cen cre ritim 2 red ma te ultu eo ilus Unc ha e3 um ot 1 arc op ae te ro s ren ch eo Nit ec ar a rin en rch ma cr a red ine cren ltu ar e m cu in d Un ar re m ltu d cu re Un lt u cu Un C – ne ota ari ae M arch n re Korarchaeota NATURE | Vol 464 | 15 April 2010 E ic Euryarchaeota Figure 2 | Phylogenetic tree of FBP aldolases/phosphatases (compare with the ribosomal proteins trees in Fig. 1). Species in red are members of earlybranching lineages of the Bacteria; the Clostridia cluster is shown in black characters. Species for which their enzymes were overproduced and studied here are underlined. The tree was constructed using the neighbour-joining algorithm. When maximum-parsimony or maximum-likelihood algorithms were used, the same groups were obtained. Bootstrap values higher than 75% are marked with dots. (Hyper)thermophiles are marked by red lineages; autotrophs by blue boxes. integrative and conjugative elements25, which may have facilitated the acquisition of the FBP aldolase/phosphatase gene. The phylogenetic tree of the enzyme (Fig. 2) largely corresponds to the phylogenetic archaeal (Fig. 1a) and bacterial (Fig. 1b) ribosomal proteins trees. Note that the phylogenetic position of Nanoarchaeota, Korarchaeota and ‘marine group I’ Crenarchaeota is currently under discussion. Both ribosomal proteins and enzyme trees clearly separate ‘marine Crenarchaeota’ from Crenarchaeota, which were suggested recently to form a new archaeal phylum26. The apparent association of members of Methanomicrobiales (Methanoculleus marisnigri and Candidatus ‘Methanoregula boonei’) with Bacteria in Fig. 2 is probably not significant. The absence of the gene in the heterotrophic Halobacteriales and in several Methanosarcinales and Methanomicrobiales (Euryarchaeota) is interpreted as loss of the gene in these derived phyla. Loss of function is expected for Nanoarchaeum equitans, which has lost the genes for all biosynthetic pathways27. The presence of the gene in some but not all major bacterial phyla can reflect either loss (in the late-branching phyla) or gain (in the early-branching lineages) since they diverged from a common ancestor. It is impossible to conclude this case here with certainty because of the low bootstrap values in the deep branches. Yet, the marked coincidence of the presence of the highly conserved gene in the deeply branching (mostly autotrophic and thermophilic) bacterial lineages as well as the distinct lineages in the phylogenetic enzyme tree support loss in the late-branching lineages rather than gain in the early bacterial lineages. Why of all phyla should these deep branches have acquired the gene? The data may, however, be interpreted differently, as indicating that the gene is archaeal specific and that very early it was transferred twice laterally from Archaea to Bacteria, followed by independent vertical transfer. Such an ambiguous situation is similar to the chimaeric nature of Thermotogales28. Whereas ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Clostridia/Firmicutes. Many of the bacterial species harbouring the gene (Fig. 2) are members of the deeply branching Clostridia/Firmicutes23. The huge impact of lateral gene transfer, often unrecognized, on prokaryote genome evolution has been impressively documented29. What makes FBP aldolase/phosphatase a peculiar aldolase? The enzyme contains four Mg21 ions required to bind the phosphate residue (C1-phosphate in FBP and C3-phosphate in dihydroxyacetone phosphate (DHAP))18 and is consequently inactivated by EDTA (80%; a typical feature of class II enzymes). The crystal structure of the Sulfolobus tokodaii enzyme18 shows that substrate binding requires the interaction of two subunits (Fig. 4a). A conserved lysine (Lys 133) and tyrosine (Tyr 348) approximate to the C2 and C4 hydroxyl groups of FBP may be essential for catalysis (Fig. 4b and Supplementar ...
Purchase answer to see full attachment

Tutor Answer

henryprofessor
School: UC Berkeley

At...

flag Report DMCA
Review

Anonymous
Excellent job

Brown University





1271 Tutors

California Institute of Technology




2131 Tutors

Carnegie Mellon University




982 Tutors

Columbia University





1256 Tutors

Dartmouth University





2113 Tutors

Emory University





2279 Tutors

Harvard University





599 Tutors

Massachusetts Institute of Technology



2319 Tutors

New York University





1645 Tutors

Notre Dam University





1911 Tutors

Oklahoma University





2122 Tutors

Pennsylvania State University





932 Tutors

Princeton University





1211 Tutors

Stanford University





983 Tutors

University of California





1282 Tutors

Oxford University





123 Tutors

Yale University





2325 Tutors