research

Focus of research

1. Structure Determination of Ether Lipids of Methanogenic and Other Archaea

2. Biosynthesis of Ether Lipids in Archaea

3. Evolution of Archaea and Bacteria

Key words: Methanogens, Archaebacteria, Ether Lipids.

Structure Determination of Ether Lipids of Archaea (Ref. S1-S16)

Major research effort in the Department of Chemistry is focused on the biochemistry of ether lipids of methanogenic and other Archaea. The studies on archaeal lipids started in 1982 following isolation, identification, and taxonomic studies of methanogens. One of the distinctive molecular feature common to all Archaea is the nature of their membrane lipids, i.e., ether lipids. Since 1982 the complete structure analyses of many kinds of diether and tetraether lipid characteristic of Archaea have been performed using strains isolated in this laboratory or those obtained from public culture collections. The structures of the major lipids of Methanobacterium thermoautotrophicum, Methanobrevibacter arboliphilus, Methanosarcina barkeri Methanothermus fervidus, Aeropyrum pernix, and Methanopyrus kandleri have been determined.

Nomenclature of Ether Lipids of Archaea : a Proposal (Ref. S2)

Although recently a number of archaeal polar lipids with ether bonds were found, there are no systematic trivial names of them.
Because the alias such as ﾞa diphytanyl ether analog of phosphatidylserineﾞ or ﾞa phosphoethanolamine derivative of diglucosyl tetraetherﾞ is too lengthy,
one is liable to use randomly his/her laboratory desgnations (for example, PNL2b or PNGL1 etc) to call them. Of course laboratory designations are merely symbols or numbers but not names. They do not have general meanings to be understood by other workers.
Since nearly 100 polar lipids or more from Archaea have been structurally characterized to date,
we have to have a series of trivial names of archaeal lipids. We have already proposed a nomenclature of archaeal ether lipids.

1.
Archaeol----2,3-di-O-alkyl sn-glycerol diether. The fact that alkyl chains are bound at sn-2 and sn-3 positions is absolutely necessary for an archaeal ether lipid. Alkyl chains are usually isoprenoids with 20 or 25 carbons.

2.
Caldarchaeol---Two molecules of glycerol are bridged by 2 molecules of alkanediols (usually isoprenoid diols) through ether linkages which form tetraether. Ether bonds are located between the sn-2 and sn-3' positions and between sn-3 and sn-2' positions of two glycerol moieties. The name is given because caldarchaeol is a predominant core lipid of thermophilic archaebacteria (ﾞcaldﾞ means ﾞwarmﾞ).
Caldarchaeol may also include such a compound as trialkyl diglycerol tetraether which is made up from two phytanyl chains, one C40 isoprenoid and two glycerols.

3.
Isocaldarchaeol--- An isomer of caldarchaeol. Ether bonds are located between the sn-2 and sn-2' positions and between sn-3 and sn-3' positions of two glycerol moieties.

4. Archaetidic Acid and Caldarchaetidic Acid---A monophosphate ester of archaeol and caldarchaeol/isocaldarchaeol. The former is a diether analog of phosphatidic acid.

5. Phosphodiester of Archaetidic Acid or Caldarchaetidic Acid---Archaetidic acid or caldarchaetidic acid and an alcohol are linked through a phosphodiester bond. The alcohol may be serine, ethanolamine, glycerol, glycerophosphate, inositol and so on. These lipids are named as derivatives of archaetidic acid or caldarchaetidic acid. For instance, archaetidylserine is a diether analog of phosphatidylserine .

6. Glycosyl Archaeol and Glycosyl Caldarchaeol---A glycoside residue is bound to archaeol or caldarchaeol at a free hydroxylgroup through a glycosidic linkage.

7. Diglucosyl derivative of caldarchaetidylethanolamine should be called as diglucosyl caldarchaetidylethanolamine.

What is Heptad ? (Ref. S4, B2)

By the detailed examination of 11 polar lipids and 2 neutral core lipids which are composed of di- and tetra-ether lipids, structural regularities were formulated. The regularities are summarized as the "Heptad Concept".

1. The same kind of a polar head group found in diether lipids is present also in tetraether lipids and vice versa.

2. One polar head group found in tetraether lipids has the same stereochemical structure as that of corresponding diether lipids.

3. When polar head groups are bound separately to two glycerol moieties of tetraether lipids, these are not the same. Besides, one is a glycosyl residue, and the other is a phosphoric ester.

One group of lipid is composed of four tetraether lipids (phospholipid, glycolipid, phosphoglycolipid, and bare core lipid) and three diether lipids (phospholipid, glycolipid, and neutral lipid), which correspond to the structural halves of the tetraether lipids. It was, thus, proposed that the seven lipids are united in "a heptad of lipids". A heptad is characterized by a phosphoric ester head group. Thus, the major lipids of M. thermoautotrophicum are grouped into three (ethanolamine, serine, and inositol) heptads. Because two glycolipids and two neutral lipids are common to all heptads, three heptads contains 13 lipids.

What does Mean the Heptads ? (Ref. S4, B2)

The heptad concept not only summarizes structural regularities of di- and tera-ether lipids, but also implies biosynthetic relationship between diether and tetraether lipids.
The biosynthesis of tetraether polar lipids might occur by head-to-head condensation of diether polar lipids. This possible pathway postulates that the condensation between molecules of the diether type of phospholipids, glycolipid or archaeol yields the tetraethether type of phospholipids, glycolipid, phosphoglycolipids, or free caldarchaeol. The first two rule of the heptad concept are obviously logical consequences from our model of tetraether lipid biosynthesis. Therefore, the heptad hypothesis is not only based on the structural regularity but also includes the mechanism of tetraether lipid biosynthesis.

Four Characteristic Features of Archael Ether Lipids

1. Ether linkage : Alkyl chains are bound to glycerol moiety by ether linkages, in contrast to ester linkages in other organisms (Bacteria and Eucarya).

2. Isoprenoid chain : Hydrocarbon chains are isoprenoid (mainly phytanyl and bisphytanediyl) chains, which is different from the almost straght chain fatty acids.

3. Enantiomeric configuration : The alkyl chains are etherified at the sn-2 and sn-3 positions of glycerol moiety, which is the enantiomeric configuration of bacterial and eukaryal ester lipids.

4. Tetraether bridged form lipid : In some Archaea tetraether lipids are present.

Polar Lipids as a Chemotaxonomic Marker (Ref. T1-T3)

Based on the results of the structural studies, we developed a method of lipid component parts analysis to determine an outline of lipid composition of many species of methanogens. The distribution of lipid component parts in nearly half of all methanogen species has been analyzed by this method. This method has been proved to be useful as a tool for taxonomic identification of the methanogens, and co-operative studies with laboratories in U.S. on a new taxonomy of methanogens are carried out.

What is the Component Parts Analysis ? (Ref. T1-T3)

In these studies, a great variety of lipids have been shown, i.e., five kinds of core lipids (alkyl glycerol ethers), seven or more polar head groups and at least three sugar moieties have been identified. On the basis of the diversity of polar lipid structures of methanogens, it was considered that polar lipid composition may be employed for methanogen taxonomy. In spite of the simplicity of TLC procedure, TLC is essentially less informative regarding the chemical structures of lipids, and TLC patterns could be compared if they were carried out only in a standardised system (using standardised solvent systems and a certain batch of TLC plates). Although the best way to show the relationship between lipids is to compare their complete structures, the complete structural determination of a lipid is a laborious and time-consuming task that is not suitable for rapid taxonomic work. We intended to develop a method that is more informative in lipid structure than TLC and less time-consuming in analysis than complete structural determination, i.e., the analysis of the component parts of polar lipids in the total lipids. The combination of the occurrence of these component parts should give a significant information on a chemotaxonomic importance of polar lipids.

The ether core lipids, phospholipid-polar head groups, and glycolipid-sugar moieties (component parts of polar lipids) from methanogens were analysed qualitatively at the total lipid level, without separation of lipids after extraction, and the results were cumulated.
Archaeol, caldarchaeol, macrocyclic archaeol, two kinds of hydroxyarchaeol, H-shaped caldarchaeol, and unsaturated archaeols were found as core lipids; myo-inositol, ethanolamine, serine, aminopentanetetrols, glycerol, and choline were identified as phospholipid-polar head groups; and glucose, galactose, mannose and N-acetylglucosamine as glycolipid-sugars in methanogens as a whole.
The distribution of these component parts, regardless of their arrangement in the lipid molecules, was characteristic of methanogen taxonomic groups at a family or genus level, and, therefore, coincided with the classification based on the 16S rRNA analysis. This shows that lipid component parts could become a new chemotaxonomic marker, which utilizes more lipid-structure-oriented information than a TLC pattern.
(Table 1, Table 2, Table 3)

Application of Lipid Analysis to Ecological Studies of Methanogenic Archaea (Ref. E1-E5)

Lipid is localized in a single cell membrane in methanogenic archaea as prokaryotes. Because cells of methanogens range in an almost similar sizes regardless of species, it can be considered that the cell surface area is also constant regardless of species. The cell number or biomass of methanogens is, therefore, proportional to the amount of ether lipid of methanogens. Since ether lipid is different in chemical nature from ester lipid of Bacteria and Eucarya, one can easily estimate the amount of methanogen ether lipid in the presence of ester lipid of coexisting Bacteria/Eucarya in a natural sample. By mild alkaline treatment of total lipid extracted from a natural sample, ether lipid can be separated from alkaline-labile ester lipid. The resultant ether lipid is converted to a derivative of uv-absorbing or fluorescent compound, and then analyzed by an HPLC in high sensitivity. We applied this method to samples from sludges of anaerobic sewage digestors, soils from paddy fields in Japan, and sediment samples from Tokyo Bay, and estimated cell numbers and biomass of methanogens in those ecosystems.

Biosynthesis of Polar Ether Lipid in Archaea (Ref. B1-B12,D1)

As the outlines of the ether lipids of the methanogens have been elucidated, the studies on the biosynthesis of ether lipids and enzymes involved in the pathway have started in 1993.

1. One of the enzymes involved in the archaeal lipid biosynthesis, sn-glycerol-1-phosphate dehydrogenase has been purified and characterized. This enzyme is responsible to the formation of sn-glycerol-1-phosphate backbone of the enantiomeric configuration of archaeal polar lipids, which is one of the most characteristic features of Archaea. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity with that of NAD(P)-linked sn-glycerol-3-phosphate dehydrogenase of Escherichia coli which catalyzes the formation of sn-glycerol-3-phosphate backbone of bacterial phospholipids. Database search revealed that sn-glycerol-1-phosphate dehydrogenase shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase and sn-glycerol-1-phosphate dehydrogenase does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. The model predicted that the enzyme selectively uses the pro-R hydrogen of the NADH. Transfer of pro-R hydrogen was experimentally confirmed.
It was shown by Poulter and his coworkers that sn-glycerol-1-phosphate was etherified with geranylgeranyl pyrophosphate to form unsaturated archaetidic acid.

2. We found the activity which catalyzed transfer of CMP moiety from CTP to unsaturated archaetidic acid (CDP-unsaturated archaeol synthase) in the membrane fraction of Methanothermobacter thermautotrophicus. CDP-unsaturated archaeol is expected as an important intermediate of biosynthesis of various kinds of polar lipids in Archaea. Although two enzymes that catalyze the formation of ether bonds in the lipids are specific to sn-glycerol-1-phosphate, CDP-archaeol synthase is not stereospecific, even nor ether lipid-specific. The enzyme prefers geranylgeranyl group-containing archaetidic acid to saturated lipids. CDP-archaeol is converted to archaetidylserine by the action of archaetidylserine synthase, which was found in the membrane fraction of M. thermoautotrophicus. Archaeal archaetidylserine synthase is a member of phosphatidylserine synthase II(Bacillus subtilis) subfamily according to the complete genome information.

3. A Blast search with archaetidylserine synthase from M.thermautotrophicus as a query revealed that archaetidylglycerol synthase and archaetidylinositol synthase also belong to the same enzyme family "CDP alcohol phosphatidyltransferase family".

Hypothesis for the Differentiation of Archaea and Bacteria by the Different Enantiomers of Glycerophosphate in Their Membrane Phospholipids (Ref. D1)

1. To interpret significance and reason for existence of the different enantiomers of glycerophosphate in the cells of Archaea and Bacteria, we proposed a hypothesis that ancestors of Archaea and Bacteria happened to differentiate by adoption of different enantiomers of glycerophosphate in their membrane phospholipids when first cells were enclosed by the membranes.
Because the enzymes with an opposite stereospecificity have quite different sequences as shown above in the case of sn-glycerol-1-phosphate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase, it seems quite unlikely that the stereospecificity of the glycerophosphate-forming enzyme of Archaea or Bacteria could be reversed.

2. It is likely that the enantiomer of glycerophosphate of membrane phospholipids was hardly able to be replaced by another enantiomer in one organism once the stereospecificity of the enzyme had been established in an organism. This means that the origin of the stereochemical structures of glycerophosphate in Archaea and Bacteria could be retrospected to the time of the differentiation of the two groups.

3. Because glycerophosphate is the first stereospecific precursor of phospholipid biosynthesis in organisms of both domains, the stereostructure of glycerophosphate prescribes the stereostructure of the derived lipids, and difficulty of changeover from one stereoisomer of glycerophosphate to the other in an organism is multiplied by the number of stereospecific enzymes involved in the synthesis of polar lipids. Thus it seems likely that the established stereospecificity of the enzymes on the pathway is kept almost permanently by heredity.

4. It is noteworthy that glycerophosphate constitutes the backbone of membrane phospholipids. A cell membrane is of special significance in differentiation of cells of Archaea and Bacteria because membrane structure is absolutely essential for a cell. A cell is a small room separated from outside environment by a partition (membrane). A cell membrane defines cell; no membrne --no cell. A cell was first born when soluble metabolites, genetic machineries and biological catalysts etc of a cell was enclosed by a membrane (probably made of phospholipids). A membrane made of phospholipids with either enantiomer of glycerophosphate might separately insulate intracellular process (metabolism) and it should have established a cell, which would be an ancestor of the either domains of life (Archaea and Bacteria). Substantial metabolism without cells has been suggested to be present as a form such as surface metabolism on pyrite proposed by Wachtershauser before cells were enclosed by lipid membranes, since most basic biochemical features were shared by Archaea and Bacteria.

5. At the chemical evolution stage, both enantiomers of glycerophosphate would be synthesized as phospholipid precursors when stereospecificity of a biosynthetic catalyst would not be established. In later time two types of catalysts or enzymes specific for either stereoisomer became evolved, and the membrane would be composed of either enantiomer of glycerophosphate. Thus separation of Archaea and Bacteria, two domains of life, might be caused by cellularization by membranes with two enantiomeric lipids synthesized with sn-glycerol-1-phosphate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase evolved from different enzymes, respectively.

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[update 2. 28, 2006 edited by Dept.of Chemistry]

1.	Archaeol----2,3-di-O-alkyl sn-glycerol diether. The fact that alkyl chains are bound at sn-2 and sn-3 positions is absolutely necessary for an archaeal ether lipid. Alkyl chains are usually isoprenoids with 20 or 25 carbons.
2.	Caldarchaeol---Two molecules of glycerol are bridged by 2 molecules of alkanediols (usually isoprenoid diols) through ether linkages which form tetraether. Ether bonds are located between the sn-2 and sn-3' positions and between sn-3 and sn-2' positions of two glycerol moieties. The name is given because caldarchaeol is a predominant core lipid of thermophilic archaebacteria (ﾞcaldﾞ means ﾞwarmﾞ). Caldarchaeol may also include such a compound as trialkyl diglycerol tetraether which is made up from two phytanyl chains, one C40 isoprenoid and two glycerols.
3.	Isocaldarchaeol--- An isomer of caldarchaeol. Ether bonds are located between the sn-2 and sn-2' positions and between sn-3 and sn-3' positions of two glycerol moieties.
4.	Archaetidic Acid and Caldarchaetidic Acid---A monophosphate ester of archaeol and caldarchaeol/isocaldarchaeol. The former is a diether analog of phosphatidic acid.
5.	Phosphodiester of Archaetidic Acid or Caldarchaetidic Acid---Archaetidic acid or caldarchaetidic acid and an alcohol are linked through a phosphodiester bond. The alcohol may be serine, ethanolamine, glycerol, glycerophosphate, inositol and so on. These lipids are named as derivatives of archaetidic acid or caldarchaetidic acid. For instance, archaetidylserine is a diether analog of phosphatidylserine .
6.	Glycosyl Archaeol and Glycosyl Caldarchaeol---A glycoside residue is bound to archaeol or caldarchaeol at a free hydroxylgroup through a glycosidic linkage.
7.	Diglucosyl derivative of caldarchaetidylethanolamine should be called as diglucosyl caldarchaetidylethanolamine.

1.	The same kind of a polar head group found in diether lipids is present also in tetraether lipids and vice versa.
2.	One polar head group found in tetraether lipids has the same stereochemical structure as that of corresponding diether lipids.
3.	When polar head groups are bound separately to two glycerol moieties of tetraether lipids, these are not the same. Besides, one is a glycosyl residue, and the other is a phosphoric ester.

1.	Ether linkage : Alkyl chains are bound to glycerol moiety by ether linkages, in contrast to ester linkages in other organisms (Bacteria and Eucarya).
2.	Isoprenoid chain : Hydrocarbon chains are isoprenoid (mainly phytanyl and bisphytanediyl) chains, which is different from the almost straght chain fatty acids.
3.	Enantiomeric configuration : The alkyl chains are etherified at the sn-2 and sn-3 positions of glycerol moiety, which is the enantiomeric configuration of bacterial and eukaryal ester lipids.
4.	Tetraether bridged form lipid : In some Archaea tetraether lipids are present.

1.	One of the enzymes involved in the archaeal lipid biosynthesis, sn-glycerol-1-phosphate dehydrogenase has been purified and characterized. This enzyme is responsible to the formation of sn-glycerol-1-phosphate backbone of the enantiomeric configuration of archaeal polar lipids, which is one of the most characteristic features of Archaea. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity with that of NAD(P)-linked sn-glycerol-3-phosphate dehydrogenase of Escherichia coli which catalyzes the formation of sn-glycerol-3-phosphate backbone of bacterial phospholipids. Database search revealed that sn-glycerol-1-phosphate dehydrogenase shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase and sn-glycerol-1-phosphate dehydrogenase does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. The model predicted that the enzyme selectively uses the pro-R hydrogen of the NADH. Transfer of pro-R hydrogen was experimentally confirmed. It was shown by Poulter and his coworkers that sn-glycerol-1-phosphate was etherified with geranylgeranyl pyrophosphate to form unsaturated archaetidic acid.
2.	We found the activity which catalyzed transfer of CMP moiety from CTP to unsaturated archaetidic acid (CDP-unsaturated archaeol synthase) in the membrane fraction of Methanothermobacter thermautotrophicus. CDP-unsaturated archaeol is expected as an important intermediate of biosynthesis of various kinds of polar lipids in Archaea. Although two enzymes that catalyze the formation of ether bonds in the lipids are specific to sn-glycerol-1-phosphate, CDP-archaeol synthase is not stereospecific, even nor ether lipid-specific. The enzyme prefers geranylgeranyl group-containing archaetidic acid to saturated lipids. CDP-archaeol is converted to archaetidylserine by the action of archaetidylserine synthase, which was found in the membrane fraction of M. thermoautotrophicus. Archaeal archaetidylserine synthase is a member of phosphatidylserine synthase II(Bacillus subtilis) subfamily according to the complete genome information.
3.	A Blast search with archaetidylserine synthase from M.thermautotrophicus as a query revealed that archaetidylglycerol synthase and archaetidylinositol synthase also belong to the same enzyme family "CDP alcohol phosphatidyltransferase family".

1.	To interpret significance and reason for existence of the different enantiomers of glycerophosphate in the cells of Archaea and Bacteria, we proposed a hypothesis that ancestors of Archaea and Bacteria happened to differentiate by adoption of different enantiomers of glycerophosphate in their membrane phospholipids when first cells were enclosed by the membranes. Because the enzymes with an opposite stereospecificity have quite different sequences as shown above in the case of sn-glycerol-1-phosphate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase, it seems quite unlikely that the stereospecificity of the glycerophosphate-forming enzyme of Archaea or Bacteria could be reversed.
2.	It is likely that the enantiomer of glycerophosphate of membrane phospholipids was hardly able to be replaced by another enantiomer in one organism once the stereospecificity of the enzyme had been established in an organism. This means that the origin of the stereochemical structures of glycerophosphate in Archaea and Bacteria could be retrospected to the time of the differentiation of the two groups.
3.	Because glycerophosphate is the first stereospecific precursor of phospholipid biosynthesis in organisms of both domains, the stereostructure of glycerophosphate prescribes the stereostructure of the derived lipids, and difficulty of changeover from one stereoisomer of glycerophosphate to the other in an organism is multiplied by the number of stereospecific enzymes involved in the synthesis of polar lipids. Thus it seems likely that the established stereospecificity of the enzymes on the pathway is kept almost permanently by heredity.
4.	It is noteworthy that glycerophosphate constitutes the backbone of membrane phospholipids. A cell membrane is of special significance in differentiation of cells of Archaea and Bacteria because membrane structure is absolutely essential for a cell. A cell is a small room separated from outside environment by a partition (membrane). A cell membrane defines cell; no membrne --no cell. A cell was first born when soluble metabolites, genetic machineries and biological catalysts etc of a cell was enclosed by a membrane (probably made of phospholipids). A membrane made of phospholipids with either enantiomer of glycerophosphate might separately insulate intracellular process (metabolism) and it should have established a cell, which would be an ancestor of the either domains of life (Archaea and Bacteria). Substantial metabolism without cells has been suggested to be present as a form such as surface metabolism on pyrite proposed by Wachtershauser before cells were enclosed by lipid membranes, since most basic biochemical features were shared by Archaea and Bacteria.
5.	At the chemical evolution stage, both enantiomers of glycerophosphate would be synthesized as phospholipid precursors when stereospecificity of a biosynthetic catalyst would not be established. In later time two types of catalysts or enzymes specific for either stereoisomer became evolved, and the membrane would be composed of either enantiomer of glycerophosphate. Thus separation of Archaea and Bacteria, two domains of life, might be caused by cellularization by membranes with two enantiomeric lipids synthesized with sn-glycerol-1-phosphate dehydrogenase and sn-glycerol-3-phosphate dehydrogenase evolved from different enzymes, respectively.