DI- AND TETRA-ALKYL ETHER LIPIDS OF THE ARCHAEA

1. Basic Chemistry

The Archaea represent one of the three primary kingdoms or domains of living organisms. The alternative name 'archaebacteria' is considered by some to be redundant. Archaea are single celled, lacking a nuclear membrane and having a low deoxyribonucleic acid content. They include thermophiles, halophiles and acidophiles; collectively, these are termed 'extremophiles'. Some believe that they resemble the dominant organisms in the primeval biosphere. Many of the species are methanogenic, including those found in low temperature environments. Four archaeal phyla are recognized, the euryarchaeota, crenarchaeota, korarchaeota and nanoarchaea. These exist in extreme habitats, including hot springs and waters with high salt, alkali or acid conditions, but more recently it has become apparent that many such organisms exist in mild environmental conditions, and indeed it is now suggested that these may constitute up to 20% of the oceanic biomass.

The lipids of these organism are now known to contain many unique and characteristic polar lipids, based on 2,3-dialkyl-sn-glycerol backbones, i.e. the stereochemistry is the opposite of that found in the two other primary kingdoms, Bacteria (eubacteria) and Eucarya (eukaryotes).

structures - archaeol and caldarchaeol

The alkyl groups are isoprenoid in nature, and the simplest molecules of this type are derivatives of 2,3-diphytanyl-O-sn-glycerol (archaeol), i.e. with two C₂₀ isoprenoid units attached to positions sn-2 and sn-3 of glycerol by ether linkages (sometimes designated C₄₀ ether lipids). Many other isoprenoid groups are found linked in this way, including macrocyclic diethers. Generally, the alkyl chains are saturated, but forms with double bonds in various positions have been found in a few species of Archaea.

In addition, series of tetraethers with strikingly different molecular architecture have been discovered as core lipids of the Archaea. These molecules have one or two polar head groups, which need not be the same, with the 2,3-sn-glycerol moieties linked by two C₄₀ alkyl components that are also isoprenoid in nature (C₈₀ ether lipids). Thus, caldarchaeol (so-called because it is the predominant form in thermophilic Archaea) has two C₄₀ isoprenoid units linked from position 2 to position 3’ and from position 3 to position 2’, while in isocardarcheol, they are linked from position 2 to position 2’ and from position 3 to position 3’ of the two sn-glycerol moieties.

Some lipids of this type have both methyl branches and one to four cyclopentane rings, while the crenarchaeota may have an additional cyclohexane ring in the alkyl chains. There appear to be increasing proportions of cyclopentane rings in lipids from thermoacidophilic species with increasing environmental temperatures. Yet other lipids have of this type have carbon-carbon links between the chains, forming an ‘H’-shape. While a very large number of biphytanyl structures of this type could potentially exist, only nine have so far been identified in living organisms; others may exist as related molecules with up to eight cyclopentyl rings have been observed in naphthenate deposits in crude oil processing. As an example, the structure illustrated forms part of the tetra-ether core of crenarchaeol from hyper-thermophilic organisms of the Crenarchaeota.

part structure of crenarchaeol

Formula of calditol As an alternative to the simple glycerol component, calditol from Sulfolobus solfaricus contains 2-hydroxymethyl-1,2,3,4,5-pentahydroxy-cyclopentane linked via an ether bond to the glycerol moiety at position sn-1. In other species, the alkyl groups are linked to tetritol.

While these core lipids are often found in the free state in organisms, more often they are completed by having a variety of polar head groups. These exist as both as phospho- and glycolipids (and as a combination of both), and as sulfated forms of these. Most of the polar head groups of phospholipids are similar to those of organisms of the other primary kingdoms and include ethanolamine, L-serine, glycerol, myo-inositol, and even choline in phosphodiester-linkage. The glycolipids comprise mainly glucosyl and gentiobiosyl (β-D-glucosyl-(1→6)-β-D-glucosyl) units linked to the core alkylglycerols. However, in some species of Archaea there are some unique polar groups, such as di- and trimethylaminopentanetetrols, glucosaminyl-myo-inositol and glucosyl-myo-inositol. New structures are still being discovered.

The main lipids in Archaea

The simplest lipids of this type are based upon the archaeol backbone, so that archaetidic acid is the monophosphate ester of archeol and is the equivalent of phosphatidic acid from eukaryotes, while archaetidylethanolamine is analogous to phosphatidylethanolamine, for example. Indeed, most of the conventional phospholipids have archaeal equivalents, including an analogue of cardiolipin. Similarly, gentiobiosyl archaeol could be considered as an analogue of the diglycosyldiacylglycerols found in higher plants.

Other such lipids, which may lack conventional equivalents, include archaetidylglycerosulfate, archaetidylglycerophosphate methyl ester and triglycosyl archaeol lipids. It appears that aminolipids and glycolipids containing pH-sensitive β-D-galactofuranosyl units are common in the methanogens, but they are absent from thermophiles. Streptococcus species contain dimeric lipids, termed 'glucopyronosy- and kojibiosyl-cardiolipins', while Halobacterium salinarum contains similar lipids with sulfo-tri- and diglycosyl-diethers esterified to the phosphate group of phosphatidic acid. However, the term ‘glycocardiolipin’ is misleading, as a glucose unit takes the place of the central glycerol of cardiolipin.

The lipids based on the caldarchaeol and other tetra-ether cores are much more complicated. Often only one of the glycerol moieties is attached to a polar moiety, so caldarchaetidic acid is the monophosphate ester of caldarchaeol. However, more often both glycerols are linked to polar moieties, and these are always different, e.g. glycosyl caldarchaetidylserine contains a glycosyl moiety at one end of the molecule and a serine phosphate at the other. Thus, the extensively studied species Methanobacterium thermoautotrophicum contains four phospholipids (archaetidic acid, archaetidylethanolamine, archaetidylserine, archaetidyl-myo-inositol) and one glycolipid (gentiobiosyl archaeol) based on archaeol, together with four phospholipids (caldarchaetidic acid, caldarchaetidylethanolamine, caldarchaetidylserine, caldarchaetidyl-myo-inositol), one glycolipid (gentiobiosyl caldarchaeol) and three mixed glyco-phospholipids (gentiobiosyl caldarchaetidylethanolamine, gentiobiosyl caldarchaetidylserine, gentiobiosyl caldarchaetidyl-myo-inositol) based on caldarchaeol.

Different archaeal species can contain distinctive variants on the basic structures, which are proving useful for taxonomic purposes. Also, residues of archaeal ether lipids can survive well in rocks and sediments, and can serve as markers for the archaea in general and for particular organisms over geological time spans.

It is not always recognised that Archaeal species can contain appreciable amounts of lipids containing conventional fatty acids. Most of these are not linked by ester bonds, so may be in unesterified form or as amide-linked components of amino-lipids, such as ornithine lipids, sphingolipids or proteolipids. However, in Methanothermus fervidus, esterified fatty acids amounted to 89.0% of the total phospholipid side chains. None of these lipids appear to have been adequately characterised as yet.

2. Archaeal Lipids in Membranes

Diether phospholipids resemble the more conventional diacyl phospholipids from eukaryotes in many aspects of their physical properties, and in particular they have an ability to form bilayer membrane structures. On the other hand, tetra-ether polar lipids can span the membranes of the organisms to form in effect a membrane monolayer. Physical chemical methods, such as freeze fracturing, are not able to separate the two leaves of the bilayer, for example. In aqueous solution, the bipolar ether lipids especially form remarkably stable liposomes of different sizes (uni- and multi-lamellar) and membrane packing densities, a property of potential value as carriers of therapeutic agents or as adjuvants of drugs and vaccines.

Scottish thistle The complex archaeal lipids are distributed asymmetrically in membranes. A study of the distribution of lipids between the inner and outer leaflets of the membrane of Methanobacterium thermoautotrophicum has demonstrated that a high proportion of the gentiobiose units of both the di- and tetra-ether lipids are exposed on the outer aspect of the cells, where inter-glycosyl hydrogen bonding may assist in stabilizing the membrane structure. Similarly, much of the gentiobiose unit of gentiobiosyl caldarchaetidylethanolamine is on the outer surface with the phosphoethanolamine unit inside, although most of the archaetidylethanolamine (diether) is in the outer leaflet of the membrane bilayer. The phosphoserine and phosphoinositol residues of both diether and tetraether polar lipids are mainly oriented towards the cytoplasmic surface of the membrane.

Ether lipids are much more stable to chemical attack via oxidation or acid/base treatment than acyl lipids, and there is increasing evidence that they have a major role in archaeal membranes in enabling the organisms to tolerate extremes of temperature, salt concentrations and pH. The addition of cyclic structures such as five-membered rings to the trans-membrane portion of the lipids appears to be an adaptation to high temperatures, conferring enhanced membrane packing and reduced fluidity properties, and strengthens the hydrogen bonding at the membrane surface. The presence of a covalent bond between the alkyl chains in H-shaped tetraethers may reinforce the strength of the membrane. For example, halophiles can thrive at salinities greater than 20-25%, while the optimal growth temperature for many thermophiles is 80°C, and some have survived a temperature as high as 120°C. Acidophiles are able to withstand a pH of zero and below. It has been demonstrated that the tetra-ether membrane monolayers especially have a limited permeability for protons even at the higher growth temperatures that have been observed. It appears that Archaea adjust the composition of their membrane lipids to maintain their proton permeability within a narrow range. Membranes containing tetra-ether lipids are also able to withstand high concentrations of metal ions and pH gradients that approach 5 pH units.

In mitochondria and bacteria, there is a tight association between cardiolipin and cytochrome c oxidase, and this is also true for the cardiolipin analogue in Archaea, suggesting that this is a truly universal lipid–protein interaction.

3. Biochemistry

The unique chirality of the glycerol molecule in these lipids is a consequence of the specificity of the enzyme that reduces dihydroxy acetone phosphate, i.e. the product is sn-glycerol-1-phosphate rather than sn-glycerol-3-phosphate, as in bacteria and eukaryotes.

Synthesis of sn-glycerol-1-phosphate

In eukaryotes, there are a number of routes to the generation of sn-glycerol-3-phosphate, including via glycolysis, in addition to via D-glyceraldehyde-3-phosphate as illustrated (see our web page on ether lipids). Dihydroxyacetone phosphate (DHAP) is a key intermediate, which in Archaea is converted to sn-glycerol-1-phosphate by an NADH-dependent reduction a by G1P-dehydrogenase. The evolutionary significance of this is a matter of debate, with various experts ranking the deviation in these pathways at different points in the divergence of Archaea and Bacteria from a primitive ancestral cell. Comparisons of other enzymes in the biosynthesis of lipids in also relevant to this debate, which I prefer to leave to the experts (see the reading list below).

The isoprenoid chains are synthesised by a mechanism that appears to be very similar to the classical mevalonic acid pathway (see our web page on cholesterol biosynthesis). For example, the key enzyme HMG-CoA reductase from Sulfolobus solfataricus showed more than 40% similarity to eucaryal homologues. Overall, some of the relevant enzymes in the synthesis of the isoprenoid chains are closely related in structure to those in bacteria while others are more similar to those of eukaryotes. In this pathway, the multifunctional enzyme geranylgeranyl diphosphate synthase catalyses the chain elongation of dimethylallyl diphosphate to form geranylgeranyl diphosphate.

structure of 2,3-digeranylgeranyl-O-sn-glycerol Ether bonds are formed by coupling the unsaturated terpenoid chains as geranylgeranyl units, first to position 3 of sn-glycerol-1-phosphate and then to position 2 to form an unsaturated archaetidic acid. Cyclopentane rings are formed by internal cyclization of the biphytanyl chains. The alkyl chains must be hydrogenated before the lipids are incorporated into membranes, but it is not certain whether this occurs before or after attachment of the polar head groups. In relation to the latter, molecular biology and gene studies have found a number of archaeal proteins with sequence similarities to members of the cytidine diphosphate (CDP)-alcohol phosphatidyltransferase family, suggesting that the biosynthesis mechanisms for the archeol serine, glycerol and inositol phospholipids resemble those for the bacterial analogues. Similarly, archaetidylethanolamine is probably synthesised by decarboxylation of archaetidylserine as in the bacterial equivalent. In the same way, archaeol glycolipids are synthesised by the transfer of glucose or gentiobiose from UDP-glucose or UDP-gentiobiose, respectively, to archaeol.

The formation of tetra-ether lipids is one of the most intriguing problems in lipid biochemistry. It is presumed that it must involve carbon-carbon bond formation between the two methyl termini of phytanyl or their precursor (unsaturated) chains. Such a reaction would be unprecedented in biochemistry, and it will be fascination to learn how the story unfolds with further research.

The AOCS Lipid Library

DI- AND TETRA-ALKYL ETHER LIPIDS OF THE ARCHAEA

1. Basic Chemistry

2. Archaeal Lipids in Membranes

3. Biochemistry

Suggested Reading