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)|
|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 Ža diphytanyl ether analog of phosphatidylserineŽ or Ža phosphoethanolamine derivative of diglucosyl tetraetherŽ 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.
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.
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 (ŽcaldŽ means ŽwarmŽ).
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)|
|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)|
|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)|
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
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
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]