Download An ACP-Independent Fatty Acid Synthesis Pathway in Archaea

An ACP-Independent Fatty Acid Synthesis Pathway in Archaea:
Implications for the Origin of Phospholipids
Jonathan Lombard, Purificación López-Garcı́a, and David Moreira*
Unité d’Ecologie, Systématique et Evolution, Université Paris-Sud, Orsay, France
*Corresponding author: E-mail: [email protected].
Associate editor: Andrew Roger
Abstract
Fatty acids (FAs) are major building blocks of membrane phospholipids in bacteria and eukaryotes. Their presumed absence in
archaea led to propose a late origin in bacteria and eukaryotes and that the last common ancestor of living organisms (the
cenancestor) was devoid of both FA and phospholipid membranes. However, small FA amounts and homologs of bacterial FA
biosynthesis enzymes are found in archaea. We have investigated the origin of these archaeal enzymes using phylogenomic
analyses of all enzymes of the main bacterial FA biosynthesis pathway. Our results suggest that modern archaea and their last
common ancestor possessed a complete pathway except for the acyl carrier protein (ACP) processing machinery, which evolved
in the bacterial lineage. This has not only implications for archaeal physiology but also opens the possibility for the presence of
ACP-independent FA synthesis in the cenancestor, which may have been endowed with FA-phospholipid membranes.
Key words: fatty acid synthesis, acyl carrier protein, archaea, early evolution, cenancestor.
Determining the evolutionary origin of archaeal FA has
implications both to envisage early evolution scenarios and
to better understand archaeal physiology. FA biosynthesis
occurs through repetitive condensation of acyl (malonylCoA) groups in a series of cyclic steps (fig. 1) catalyzed by
enzymes that may be individual (FA synthase type II [FAS-II])
or fused in one unique polypeptide (FAS-I). FAS-II is the main
FA biosynthesis pathway in most bacteria, mitochondria, and
chloroplasts (White et al. 2005), whereas FAS-I is more rare in
bacteria but predominant in the eukaryotic cytoplasm
(Schweizer and Hofmann 2004). As we were particularly interested by the presence of FAS homologs in archaea and only
individual FAS-II but not polypeptidic FAS-I homologs were
detected in the available archaeal genome sequences, we removed the FAS-I sequences from our analyses and restricted
our systematic phylogenomic analyses to the FAS-II individual
peptides. Our results support that the actual distinctive feature between archaea and bacteria is not the presence of the
FAS system itself, but the lack of the FAS-related acyl carrier
protein (ACP) processing machinery in archaea.
FAS-II Homologs in Archaea
In bacteria, several reactions supply the malonyl-ACP building
blocks necessary to FAS-II: 1) acetyl-coenzyme A (CoA) is
converted into malonyl-CoA by the acetyl-CoA carboxylase
(ACC), 2) a phosphopantetheine group from CoA is attached
by an ACP synthase (ACPS) to the apo-ACP, and 3) the
malonyl-CoA:ACP transacylase (MCAT) charges malonylCoA to holo-ACP (fig. 1). A wide diversity of archaea bear
ACC homologs of possible monophyletic origin, suggesting
their vertical inheritance from the cenancestor (Lombard and
Moreira 2011a). ACP is a peptide cofactor that covalently
binds all FA intermediates, channeling them among the
FAS-II enzymes. Except for a few unrelated species that
ß The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
Mol. Biol. Evol. 29(11):3261–3265 doi:10.1093/molbev/mss160 Advance Access publication June 19, 2012
3261
Letter
Although fatty acids (FAs) play major roles as storage molecules, secondary metabolites, and components of bacterial
and eukaryotic membrane phospholipids, little is known
about them in archaea. This reflects that, most often, archaeal
membrane phospholipids are considered to lack FA and to
carry isoprenoids instead (Boucher 2007). In fact, although
bacterial and eukaryotic phospholipids contain FA bound
to sn-glycerol-3-phosphate with ester links, in archaea, isoprenoids are linked to sn-glycerol-1-phosphate through
ether bounds (De Rosa et al. 1986; Boucher 2007). These
striking differences figured among the main cellular characteristics that supported archaea as an independent domain of
life (Woese et al. 1978).
However, experimental data show that FAs also exist in
archaea. Biochemical evidence for FA synthase machineries
(Pugh et al. 1971; Pugh and Kates 1994) and small amounts of
free FA or their derivatives (Kates et al. 1968; Langworthy
et al. 1974; Tornabene et al. 1978; Carballeira et al. 1997;
Nishihara et al. 2000) have been reported in diverse archaea.
Phospholipids bearing FA have been detected in very diverse
euryarchaeotes (Gattinger et al. 2002), and, as in bacteria and
eukaryotes, archaeal FAs are sometimes involved in protein
structure (Colella et al. 1998) and acylation (Pugh and Kates
1994). Finally, archaeal homologs of bacterial FA biosynthesis
enzymes have been reported in archaeal genomes (Peretó
et al. 2004; Iverson et al. 2012). Despite these arguments,
the significance of FA in archaea (including the last archaeal
common ancestor [LACA]) remains poorly acknowledged in
recent scientific literature (Boucher 2007; Dibrova et al. 2011).
Similarly, the assumption that the last common ancestor of
living organisms (the cenancestor) lacked FA has even served
as a supplementary argument to propose that this organism
could not synthesize phospholipids and, therefore, was
devoid of lipid membranes (Martin and Russell 2003).
Lombard et al. . doi:10.1093/molbev/mss160
MBE
FIG. 1. Archaeal homologs of the archetypical bacterial FAS-II pathway. The bacterial FAS-II enzymes are represented. Protein names and EC numbers
are indicated within boxes. Black boxes indicate the existence of archaeal homologs, potentially involved in an alternative archaeal CoA-dependent
pathway, whereas gray boxes denote bacterial enzymes absent in most archaea. ACC, acetyl-CoA carboxylase; ACP, acyl-carrier protein; ACPS, acyl
carrier protein synthase; CoA, coenzyme A; DH, beta-hydroxyacyl dehydratase; ER, enoyl reductase; KAS, beta-ketoacyl synthase; KR, beta-ketoacyl
reductase; MCAT, malonyl-coenzymeA:acyl-carrier-protein transacylase.
probably acquired ACP by independent horizontal gene
transfers (HGTs), we did not retrieve homologs of the
ACP-processing machinery (ACP, ACPS, and MCAT) in archaea. This contrasted with the presence of homologs of all
the other FAS genes in a large diversity of archaea (supplementary table S1, Supplementary Material online).
Once malonyl-ACP is produced, it is condensed with
acetyl-CoA (beta-ketoacyl synthase III [KAS III], FabH) to
yield ketoacyl-ACP, an intermediate that is sequentially reduced (beta-ketoacyl reductase [KR], FabG), dehydrated
(beta-hydroxyacyl dehydratase [DH], FabZ or FabA), and reduced again (enoyl reductase [ER], FabI). A subsequent extension with malonyl-ACP (beta-ketoacyl synthases KAS I or
II, respectively, FabB and FabF) conducts a new elongation
cycle (fig. 1). Although nonhomologous replacements exist in
some bacteria (Marrakchi et al. 2003), we have focused our
search on the most common archetypal FAS-II enzymes.
A phylogenetic analysis of the thiolase superfamily, to
which KAS I, KAS II, and KAS III belong, has recently been
published (Jiang et al. 2008). It separated this superfamily into
five clusters based on phylogenetic trees and active site architecture: archaeal thiolases, bacterial + eukaryotic thiolases,
bacterial + eukaryotic KAS I–KAS II, archaeal and eukaryotic
hydroxymethylglutaryl-CoA synthase (HMGS), and bacterial+eukaryotic KAS III-chalcone synthases (CHS). However,
this can be questioned as only one very divergent archaeal
sequence was used as an outgroup, and HMGS was recently
shown to be universal and probably present in the cenancestor (Lombard and Moreira 2011b). Thus, we carried out a
more complete analysis of the thiolase superfamily (supplementary fig. S1, Supplementary Material online). Although
these enzymes preserve strong structural similarity, primary
sequence conservation is low. However, similarities within
each group of sequences point to four different clusters
with distinctive enzymatic functions: 1) universally distributed
thiolases, 2) bacterial KAS I–KAS II, 3) bacterial KAS III–CHS,
3262
and 4) universally distributed HMGS. Thiolase and HMGS
sequences supported the separation of most bacterial and
archaeal sequences into two monophyletic groups, strongly
suggesting that the cenancestor was endowed with these two
enzymes. In contrast, KAS is widespread in bacteria and
absent in archaea. Thiolases and HMGS catalyze the removal
of an acetyl-CoA moiety from an acyl-CoA chain or the opposite condensation reaction, so the major difference
between KAS and these other enzymes is the use of substrates
linked to ACP instead of CoA. Thus, our results support that
the cenancestor was able to (de)acetylate acyl-CoA chains,
whereas the use of acyl-ACP seems to be a more recent bacterial derived character.
We obtained similar results on KR sequences that, together
with ER, belong to the short-chain dehydrogenases/reductases superfamily (Kallberg et al. 2002). As this family is very
diverse, we reconstructed independent phylogenies for KR
and ER. The KR phylogeny incorporated the closely related
PhaB acetoacetyl-CoA reductases, which are involved in polyhydroxyalkanoate (PHA) synthesis in bacteria and haloarchaea. The main enzymatic difference between PhaB and KR is
the use of CoA- instead of ACP-linked substrates, although
some enzymes are known to use both (Taguchi et al. 1999;
Ren et al. 2000). Although PhaB sequences were scattered
among KR homologs, the bacterial KR-PhaB sequences clustered according to main bacterial phyla (fig. 2). Some archaeal
sequences were dispersed among the bacterial KR-PhaB, indicating independent HGTs from bacteria. Nevertheless, a
wide diversity of uncharacterized archaeal sequences
branched at the base of the bacterial KR-PhaB cluster.
Maximum likelihood (ML) phylogenetic reconstruction of
these archaeal sequences showed at least two paralogs in
crenarchaeota (with some HGTs from them to other
groups), sisters of a basal group of euryarchaeota (supplementary fig. S2, Supplementary Material online). This result indicates the ancient presence of these genes not only in the last
MBE
Fatty Acid Biosynthesis in Archaea . doi:10.1093/molbev/mss160
FIG. 2. Schematic representation of the beta-ketoacyl reductases (KR) maximum likelihood phylogenetic tree reconstructed using 569 representative
sequences and 187 conserved sites (for the complete tree see supplementary fig. S2, Supplementary Material online). Numbers are bootstrap values.
Blue, green, and orange branches correspond to bacterial, eukaryotic, and archaeal sequences, respectively. Stars and pentagons indicate
well-characterized KR and PhaB enzymes, respectively. Triangles point to enzymes carrying both functions.
common ancestor of the crenarchaeota but likely also in
LACA. The proximity to the KR-PhaB group suggests that
these archaeal sequences may catalyze similar reactions
(probably on CoA-linked substrates, ACP being absent in archaea). As for KR, the ER phylogeny supported the monophyly of most bacterial sequences and revealed two
basal-branching ER-like paralogs in crenarchaeota, indicating
an ancient duplication in this group (supplementary fig. S4,
Supplementary Material online). HGTs from crenarchaeota to
some euryarchaeota and bacteria could be noticed, but the
presence of several basal-branching euryarchaeotal sequences
supported an ancient origin also in Euryarchaeota. With the
exception of some sequences (Halobacteriales) probably acquired by HGT from bacteria, most archaeal DH homologs
were closely related to mitochondrial DH and MaoC/PhaJ
enzymes (supplementary fig. S3, Supplementary Material
online). Mitochondrial DH acts on acyl-ACP chains, whereas
MaoC/PhaJ uses acyl-CoA substrates in PHA synthesis (Tsuge
et al. 2003; Autio et al. 2008). Paralogs could be detected in
crenarchaeota, but their monophyly was difficult to asses
because of the high sequence divergence and small number
of conserved alignment positions (112). Although the function of most archaeal enzymes remains unknown, they can
reasonably be assumed to carry out DH-type reactions on
CoA-linked substrates. In summary, our analyses suggest
that LACA bore KR, ER, and possibly DH enzymes.
Conclusion
Except for some rare cases of HGT of ACP-processing machinery enzymes from bacteria, these enzymes are completely
absent from archaea, in contrast with the presence of all other
FAS-II enzymes. Moreover, all phylogenies of FAS enzymes
support that archaeal sequences are more closely related to
bacterial enzymes acting on FAS-like substrates linked to CoA
than to enzymes using ACP-linked substrates. Thus, we propose that archaea should not be considered as completely
devoid of the FAS machinery but only of the ACP-processing
system, which seems to have specifically evolved in the bacterial lineage. Although this still requires biochemical verification, it seems likely that these enzymes account for the FA
detected in archaea (Gattinger et al. 2002). From a biochemical perspective, recent work showing that the channeling of
FA intermediates from one FAS-II enzyme to the next in the
cycle is permitted by unique ACP conformations (Ploskon
et al. 2010) raises the question of the biochemical mechanism
of this putative archaeal FAS. The potential lower efficiency of
an ACP-independent FAS could account for the small
amounts of FA detected in archaea. From an evolutionary
perspective, the presence of acyl-CoA FAS-like machineries in
both bacteria and archaea argues for the existence of a similar
pathway in the cenancestor, contrary to previous claims that
the cenancestor lacked FA (Martin and Russell 2003; Dibrova
3263
Lombard et al. . doi:10.1093/molbev/mss160
et al. 2011) or that it only bore abiotically synthesized FA
(Koonin and Martin 2005). In this context, the preeminence
of FA in present-day bacterial membranes could be explained
by the evolution of the very efficient ACP-processing machinery in this lineage, whereas archaea would have recruited
other ancestral membrane-synthesizing mechanisms (e.g., isoprenoids [Lombard and Moreira 2011b]) and relegated FA to
different cellular functions.
Materials and Methods
Similarity searches were done with basic local alignment
search tool-p (Altschul et al. 1990) or psi-BLAST for DH analyses (Altschul et al. 1997) against complete genomes available
in GenBank (supplementary table S2, Supplementary Material
online) using seed sequences retrieved from KEGG (http://
www.genome.jp/kegg). For each seed, homologs were aligned
with Muscle 3.6 (Edgar 2004). Redundant sequences and sequences out of the size range known from the literature were
removed. Preliminary trees were constructed by approximate
ML with FastTree 2.1.3 (Price et al. 2010) and refined by iterative steps to remove the sequences the most divergent from
the archaeal ones. These divergent sequences and ambiguously aligned regions were detected with Guidance (Penn
et al. 2010) and discarded using NET from the MUST package
(Philippe 1993). More accurate phylogenies were inferred by
ML on the refined sequence data sets using TREEFINDER
(Jobb et al. 2004) with the LG+D model (Le and Gascuel
2008) and four rate categories. Statistical support was assessed
by approximate bootstrap (100 replicates).
Supplementary Material
Supplementary tables S1 and S2 and figures S1–S4 are available at Molecular Biology and Evolution online (http://www
.mbe.oxfordjournals.org/).
Acknowledgments
This work was supported by the interdisciplinary programs
OPV (Origine des Planètes et de la Vie) and InTerrVie
(Interactions Terre-Vie) of the French CNRS and INSU. J.L. is
recipient of a PhD fellowship of the French Research Ministry.
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local
alignment search tool. J Mol Biol. 215:403–410.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:
3389–3402.
Autio KJ, Guler JL, Kastaniotis AJ, Englund PT, Hiltunen JK. 2008. The
3-hydroxyacyl-ACP dehydratase of mitochondrial fatty acid synthesis in Trypanosoma brucei. FEBS Lett. 582:729–733.
Boucher Y. 2007. Lipids: biosynthesis, function, and evolution. In:
Cavicchioli R, editor. Archaea, molecular and cellular biology.
Washington, DC: ASM Press. p. 341–353.
Carballeira NM, Reyes M, Sostre A, Huang H, Verhagen MF, Adams MW.
1997. Unusual fatty acid compositions of the hyperthermophilic
archaeon Pyrococcus furiosus and the bacterium Thermotoga maritima. J Bacteriol. 179:2766–2768.
3264
MBE
Colella M, Lobasso S, Babudri F, Corcelli A. 1998. Palmitic acid is associated with halorhodopsin as a free fatty acid. Radiolabeling of
halorhodopsin with 3H-palmitic acid and chemical analysis of the
reaction products of purified halorhodopsin with thiols and NaBH4.
Biochim Biophys Acta. 1370:273–279.
De Rosa M, Gambacorta A, Gliozzi A. 1986. Structure, biosynthesis and
physicochemical properties of archaebacterial lipids. Microbiol Rev.
50:70–80.
Dibrova D, Makarova K, Galperin M, Koonin EV, Mulkidjanian A.
2011. Comparative analysis of lipid biosynthesis in Archaea
and bacteria: what was the structure of first membrane lipids?
Proceedings of the International Moscow Conference on
Computational Biology; 2011 Jul 21–24; Moscow (Russia):
Moscow State University.
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792–1797.
Gattinger A, Schloter M, Munch JC. 2002. Phospholipid etherlipid and
phospholipid fatty acid fingerprints in selected euryarchaeotal
monocultures for taxonomic profiling. FEMS Microbiol Lett. 213:
133–139.
Iverson V, Morris RM, Frazar CD, Berthiaume CT, Morales RL, Armbrust
EV. 2012. Untangling genomes from metagenomes: revealing an
uncultured class of marine Euryarchaeota. Science 335:587–590.
Jiang C, Kim SY, Suh DY. 2008. Divergent evolution of the thiolase
superfamily and chalcone synthase family. Mol Phylogenet Evol. 49:
691–701.
Jobb G, von Haeseler A, Strimmer K. 2004. TREEFINDER: a powerful
graphical analysis environment for molecular phylogenetics. BMC
Evol Biol. 4:18.
Kallberg Y, Oppermann U, Jornvall H, Persson B. 2002. Short-chain dehydrogenases/reductases (SDRs). Eur J Biochem. 269:4409–4417.
Kates M, Wassef MK, Kushner DJ. 1968. Radioisotopic studies on the
biosynthesis of the glyceryl diether lipids of Halobacterium cutirubrum. Can J Biochem. 46:971–977.
Koonin EV, Martin W. 2005. On the origin of genomes and cells within
inorganic compartments. Trends Genet. 21:647–654.
Langworthy TA, Mayberry WR, Smith PF. 1974. Long-chain glycerol diether and polyol dialkyl glycerol triether lipids of Sulfolobus acidocaldarius. J Bacteriol. 119:106–116.
Le SQ, Gascuel O. 2008. An improved general amino acid replacement
matrix. Mol Biol Evol. 25:1307–1320.
Lombard J, Moreira D. 2011a. Early evolution of the biotin-dependent
carboxylase family. BMC Evol Biol. 11:232.
Lombard J, Moreira D. 2011b. Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of
life. Mol Biol Evol. 28:87–99.
Marrakchi H, Dewolf WE Jr, Quinn C, et al. (12 co-authors). 2003.
Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier
protein) reductase (FabK). Biochem J. 370:1055–1062.
Martin W, Russell MJ. 2003. On the origins of cells: a hypothesis for the
evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells. Philos
Trans R Soc Lond B Biol Sci. 358:59–83; discussion 83–55.
Nishihara M, Nagahama S, Ohga M, Koga Y. 2000. Straight-chain fatty
alcohols in the hyperthermophilic archaeon Pyrococcus furiosus.
Extremophiles 4:275–277.
Penn O, Privman E, Ashkenazy H, Landan G, Graur D, Pupko T. 2010.
GUIDANCE: a web server for assessing alignment confidence scores.
Nucleic Acids Res. 38:W23–W28.
Fatty Acid Biosynthesis in Archaea . doi:10.1093/molbev/mss160
MBE
Peretó J, López-Garcı́a P, Moreira D. 2004. Ancestral lipid biosynthesis
and early membrane evolution. Trends Biochem Sci. 29:469–477.
Philippe H. 1993. MUST, a computer package of management utilities
for sequences and trees. Nucleic Acids Res. 21:5264–5272.
Ploskon E, Arthur CJ, Kanari AL, Wattana-amorn P, Williams C, Crosby J,
Simpson TJ, Willis CL, Crump MP. 2010. Recognition of intermediate
functionality by acyl carrier protein over a complete cycle of fatty
acid biosynthesis. Chem Biol. 17:776–785.
Price MN, Dehal PS, Arkin AP. 2010. FastTree 2—approximately
maximum-likelihood trees for large alignments. PLoS One. 5:e9490.
Pugh EL, Kates M. 1994. Acylation of proteins of the archaebacteria
Halobacterium cutirubrum and Methanobacterium thermoautotrophicum. Biochim Biophys Acta. 1196:38–44.
Pugh EL, Wassef MK, Kates M. 1971. Inhibition of fatty acid synthetase in
Halobacterium cutirubrum and Escherichia coli by high salt concentrations. Can J Biochem. 49:953–958.
Ren Q, Sierro N, Witholt B, Kessler B. 2000. FabG, an NADPH-dependent
3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis
in Escherichia coli. J Bacteriol. 182:2978–2981.
Schweizer E, Hofmann J. 2004. Microbial type I fatty acid synthases
(FAS): major players in a network of cellular FAS systems.
Microbiol Mol Biol Rev. 68:501–517.
Taguchi K, Aoyagi Y, Matsusaki H, Fukui T, Doi Y. 1999. Co-expression of
3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes
induces PHA production in Escherichia coli HB101 strain. FEMS
Microbiol Lett. 176:183–190.
Tornabene TG, Wolfe RS, Balch WE, Holzer G, Fox GE, Oro J. 1978.
Phytanyl-glycerol ethers and squalenes in the archaebacterium
Methanobacterium thermoautotrophicum. J Mol Evol. 11:259–266.
Tsuge T, Taguchi K, Seiichi T, Doi Y. 2003. Molecular characterization
and properties of (R)-specific enoyl-CoA hydratases from
Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation. Int J Biol Macromol. 31:
195–205.
White SW, Zheng J, Zhang YM, Rock CO. 2005. The structural biology of
type II fatty acid biosynthesis. Annu Rev Biochem. 74:791–831.
Woese CR, Magrum LJ, Fox GE. 1978. Archaebacteria. J Mol Evol. 11:
245–251.
3265