STEROLS 4.  HOPANOIDS AND RELATED LIPIDS


STRUCTURES, 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’ (from the plant genus Hopea, from which they were isolated as components of the resin). The more complex forms are sometimes termed ‘bacteriohopanepolyols’. They were first discovered in 1973 as the compounds responsible for the alignment of the cellulose microfibrils secreted by Acetobacter xylinum.

The cyclohexane rings in hopanoids have chair-chair-chair-chair-chair conformations in comparison to sterols, which have chair-boat-chair-boat-open conformations. The resulting ring structures are planar, rigid and hydrophobic with a length corresponding to half the thickness of a membrane bilayer. Overall the molecules are amphiphilic and in that they modulate the fluidity and the permeability of phospholipid membranes, they have been called ‘sterol surrogates’.

The hopanoids are structurally highly diverse. The simplest C30 hopanoid is diploptene or hop-22(29)-ene, and this is usually found with diplopterol or hopan-22-ol.

Sturctues of hopanoids

Perhaps the most abundant hopanoid in living organisms is tetrahydroxybacteriohopane (bacteriohopanetetrol), i.e. with a distinctive 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 (bacteriohopane-32,33,34,35-tetrol), 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 (C2β, C2α or C3β) or double bonds in the ring at C6 and/or C11 with stereochemical isomers adding to the variability. The more complex of these are sometimes referred to as ‘composite structures’. Hopanon from dammar resin has the diplopterol structure but with an additional ketone group on C3. Some hopanoids are so tightly bound within organisms that they are not easily extracted for structural analysis, so it is likely that many further types will eventually be characterized.

Partial structures of some complex hopanoids

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 particular, they occur in a number of dinitrogen-fixing bacteria. However, no clear taxonomic pattern has yet emerged. In most instances, the hopanoid content of prokaryote cells is comparable to that of sterols in eukaryotic cells.

A few higher plants and some ferns, mosses and fungi contain hopanoids, although these lack the complex side-chains and have an oxygen atom at C3. Their function in eukaryotes is not known.


2. Biochemistry and Function

As with sterols, squalene is a primary precursor for the biosynthesis of the hopane skeleton. 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. Indeed, it was anomalies in the results of biosynthetic studies with hopanoids that lead to the elucidation of the MEP pathway (see our web pages dealing with biosynthesis of cholesterol and plant sterols for details). A further important distinction is that hopanoid biosynthesis does not proceed via 2,3-epoxysqualene, but rather squalene per se undergoes cyclization without migration of the methyl groups. Bacterial hopanoids lack the 3β-hydroxyl group of sterols, therefore.

Biosynthesis of hopanoids

Microbial 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). Like sterol biosynthesis, it is one of the most complex single-step processes known to biochemistry, with the formation of five ring structures, modification of thirteen covalent bonds and the generation of nine new stereochemical centres, all under precise enzymatic control.

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. Adenosylhopane (see partial structure above) is the first intermediate produced during the biosynthesis of the side chains of bacteriohopanepolyols, and this is utilized to produce the wide range of products formed in microorganisms by down-stream processing. For example, the five-carbon side-chain in the bacteriohopanetetrols is derived from the D-ribose moiety of adenosylhopane after loss of the adenyl group. However, relatively little is known of the enzymology of these reactions.

The presence of the 3-oxygen atom in plant hopanoids suggests that these are formed via a 2,3-epoxysqualene intermediate.

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. They are reportedly concentrated in detergent-resistant domains, akin to rafts, in at least one bacterial species.

It is apparent that hopanoids are essential for growth in many if not all hopanoid-producing organisms as inhibition of hopanoid biosynthesis can limit their growth markedly and selectively in comparison with other bacteria. However, some species appear to grow when hopanoid synthesis is inhibited, although they are much less resistant to environmental challenges. With cultures of single species, bacteriohopanepolyols have only been detected in cyanobacteria capable of nitrogen fixation, implicating them as markers for diazotrophy in the oceans, although there is no known mechanistic link between hopanoid production and nitrogen fixation.


3.  Geohopanoids

As the pentacyclic ring structure of hopanoids is very stable and not readily degraded, geochemists tend to look upon them as molecular fossils ('geohopanoids' or '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. However, this interpretation is controversial as such compounds have recently been detected in an anoxygenic species.

Although they are resistant they are not immune to change in the geological environment over millions of years, and stereomutation, reduction and defunctionalization occur to produce hopanols and hopanoic acids, for example, commonly with 28 to 35 carbon atoms. The pattern of the various products can have indicative importance in oil exploration, so their analysis is of great practical importance. Hopanoids are sometimes stated to be the most abundant lipids on earth, although similar claims have been made for other microbial lipids and for plant waxes.


4.  Tetrahymanol and Related Lipids

structure of tetrahymanolA 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.


5.   Analysis

Hopanoids and those derived from the bacteriohopanetetrols especially require special extraction methods because of their high polarity. Indeed some are so tightly bound that their presence in many organisms may have gone undetected. 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. This no longer appears necessary, and HPLC allied to modern mass spectrometric methods involving atmospheric-pressure chemical ionization appears to be one way forward. High temperature gas chromatography-mass spectrometry has also been applied successfully to the problem.


Suggested Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

pdf
© AOCS Credits/disclaimer Updated: May 10th, 2013 Author