STEROLS 4. HOPANOIDS AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND ANALYSIS

1. Hopanoids

Bacteria and other prokaryotic organisms such as blue-green algae do not in general contain any of the conventional sterols found in plants and animals, but rather many species have related molecules, i.e. pentacyclic triterpenoids, based on a hopane skeleton with a cyclopentane E-ring and termed ‘hopanoids’. They have been termed ‘sterol surrogates’. The five rings in hopanoids have chair-chair-chair-chair-chair conformations in comparison to sterols, which have chair-boat-chair-boat-open conformations. The simplest C₃₀ hopanoid is diploptene or hop-22(29)-ene and this is usually found with diplopterol or hopan-22-ol.

hopanoids - basic structures

The hopanoids are structurally highly diverse. Perhaps the most abundant hopanoid in nature is tetrahydroxybacteriohopane (bacteriohopanetetrol), i.e. with a five-carbon terminal side-chain linked by a carbon-carbon bond to the isopropyl group of the hopane framework and with four hydroxyl groups. Many forms of this are known in which the terminal hydroxyl (C₃₅) group is substituted, for example with a glycosidic or ether linkage to glucosamine, adenosine or ribose. In some species, the polyhydroxy side-chain can be acylated, and Frankia species contain the phenylacetate monoester of tetrahydroxybacteriohopane, for example. Further hydroxylated forms are known, and penta- and hexaols and amino-polyols occur in some genera. Some of these have additional methyl groups or double bonds in the ring, while stereochemical isomers add to the variability.

Scottish thistle Hopanoids are most abundant in aerobic bacteria (methanotrophs, heterotrophs and cyanobacteria), but they also occur in some anaerobic bacteria, but not in Archaea or eukaryotes. In most instances, the hopanoid content of prokaryote cells is comparable to that of sterols in eukaryotic cells. The C₃₀ hopanoids are believed to have very similar functions to those of sterols in the membranes of animals and plants in that they modulate the fluidity of membranes by interacting with their complex lipid components to increase the degree of order or rigidity. They are important in adjusting membrane permeability, including the diffusion of oxygen, and in adaptation to extreme conditions. However, they may differ from sterols in their ability to direct vesicle formation. The hopanoid polyols share some of the properties of the C₃₀ compounds, although they may also have similar functions to those of glycolipids in eukaryotic organisms. For example, in Frankia species, most of the lipids in the membrane barrier that prevents diffusion of oxygen into the nitrogen-fixing cells are hopanoids. Hopanoids have been located in the plasma membrane and outer membranes of Gram-negative bacteria, and in the outer membrane and thylakoid membrane of cyanobacteria.

It is apparent that hopanoids are essential for growth in hopanoid-producing organisms as inhibition of hopanoid biosynthesis limits their growth markedly and selectively in comparison with other bacteria.

As the pentacyclic ring structure of hopanoids is very stable and not readily degraded, geochemists tend to look upon them as molecular fossils (‘homohopanoids’) that serve as biological markers for particular organisms in geological formations from recent sediments to petroleum deposits and rocks. Different bacterial genera possess characteristic hopanoid distributions. For example, 2α-methylhopanes that are characteristic of the photosynthetic cyanobacteria have been found in 2.7 billion year old shales in Australia, i.e. from a period long before the atmosphere was oxidizing (this interpretation is controversial). Although they are resistant they are not immune to diagenetic change in the geological environment, and stereomutation and other reactions occur producing hopanols and hopanoic acids, for example. Hopanoids are sometimes stated to be the most abundant lipids on earth, although a similar claim has been made for plant waxes.

As with sterols, squalene is a primary precursor for the biosynthesis of the hopane skeleton. However, in this instance, the process does not proceed via 2,3-epoxysqualene, but rather squalene per se undergoes cyclization without migration of the methyl groups. Hopanoids lack the 3β-hydroxyl group of sterols, therefore. Biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the intermediates in squalene biosynthesis, is via the ‘non-mevalonate’ or ‘2C-methyl-D-erythritol-4-phosphate (MEP)’ pathway (see our web pages dealing with biosynthesis of cholesterol and plant sterols for comparison).

Biosynthesis of hopanoids

Hopanoids are synthesized by a squalene-hopene cyclase that is quite distinct from the squalene epoxide cyclase. Studies of the crystal structure of the former suggest that the active site is located in a large central cavity of a size and shape to bind squalene in the necessary conformation. The cavity is surrounded by loops containing aromatic residues, which may stabilize the putative ionic intermediates. It is believed that cyclization begins with a reaction in which a carbon-carbon double bond is protonated via an aspartate residue at the top of the cavity. Then rings A and probably B are formed in a concerted manner before rings C and D are fashioned in ring closure reactions. Finally, the ring E is formed and the carbocation at C-22 is deprotonated to form hopene (or reacts with the elements of water to form diplopterol). All the reactions are under precise enzymatic control to generate the nine new stereochemical centres.

Addition of methyl groups to form 2β- and 3β-methyl hopanoids probably occurs after synthesis of the hopanoid rings, with S-adenosylmethionine as the methyl donor. The five-carbon side-chain in the bacteriohopanetetrols is derived from D-ribose via the non-oxidative pentose phosphate pathway. However, relatively little is known of the enzymology of these reactions.

2. Tetrahymanol and Related Lipids

structure of tetrahymanol A number of pentacyclic compounds related to the hopanoids are known that are derived from the gammacerane skeleton in which the E-ring is six-membered. The most important of these is tetrahymanol (gammaceran-21α-ol), which was first isolated from the ciliate protozoan Tetrahymena pyriformis. It was subsequently detected in several other eukaryotic organisms, including ferns, fungi and protozoa, before it was found in prokaryotes, such as the purple non-sulfur bacterium Rhodopseudomonas palustris.

Various structural variants have been found including 20α-methyltetrahymanol, 2β-methyltetrahymanol and 2β,20α-dimethyltetrahymanol, which occur with tetrahymanol per se and various hopanoids in the nitrogen-fixing, symbiotic root-nodule forming bacterium Bradyrhizobium japonicum.

Like the hopanoids, tetrahymanol is formed by a squalene-hopene cyclase, with the nature of the E-ring depending on the orientation of the terminal methyl groups during the final cyclization step. When sterols are added to cultures of Tetrahymena pyriformis, synthesis of tetrahymanol is inhibited completely, suggesting that sterols and tetrahymanol have similar functions in this organism.

Gammacerane structures have been found in sediments and other geological formations, together with the homohopanoids, where they are believed to be useful geochemical markers for ciliate protozoa.

3. Analysis

Hopanoids and those derived from the bacteriohopanetetrols especially require special extraction methods because of their high polarity. At one time, the molecular structures were simplified by removal of part of the side-chain by chemical means to facilitate analysis so much information was lost. HPLC allied to modern mass spectrometric methods involving atmospheric-pressure chemical ionization appears to be the way forward.

The Lipid Library

STEROLS 4. HOPANOIDS AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND ANALYSIS

1. Hopanoids

2. Tetrahymanol and Related Lipids

3. Analysis

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