Hopanoids

Hopanoids are a diverse subclass of triterpenoids with the same hydrocarbon skeleton as the compound hopane. This group of pentacyclic molecules therefore refers to simple hopenes, hopanols and hopanes, but also to extensively functionalized derivatives such as bacteriohopanepolyols and hopanoids covalently attached to lipid A.
The first known hopanoid, hydroxyhopanone, was isolated by two chemists at The National Gallery, London working on the chemistry of dammar gum, a natural resin used as a varnish for paintings. While hopanoids are often assumed to be made only in bacteria, their name actually comes from the abundance of hopanoid compounds in the resin of plants from the genus Hopea. In turn, this genus is named after John Hope, the first Regius Keeper of the Royal Botanic Garden, Edinburgh.
Since their initial discovery in an angiosperm, hopanoids have been found in plasma membranes of bacteria, lichens, bryophytes, ferns, tropical trees and fungi. Hopanoids have stable polycyclic structures that are well-preserved in petroleum reservoirs, rocks and sediment, allowing the diagenetic products of these molecules to be interpreted as biomarkers for the presence of specific microbes and potentially for chemical or physical conditions at the time of deposition. Hopanoids have not been detected in archaea.

Biological function

About 10% of sequenced bacterial genomes have a putative shc gene encoding a squalene-hopene cyclase and can presumably make hopanoids, which have been shown to play diverse roles in the plasma membrane and may allow some organisms to adapt in extreme environments.
Since hopanoids modify plasma membrane properties in bacteria, they are frequently compared to sterols, which modulate membrane fluidity and serve other functions in eukaryotes. Although hopanoids do not rescue sterol deficiency, they are thought to increase membrane rigidity and decrease permeability. Also, gammaproteobacteria and eukaryotic organisms such as lichens and bryophytes have been shown to produce both sterols and hopanoids, suggesting these lipids may have other distinct functions. Notably, the way hopanoids pack into the plasma membrane can change depending on what functional groups are attached. The hopanoid bacteriohopanetetrol assumes a transverse orientation in lipid bilayers, but diploptene localizes between the inner and outer leaflet, presumably thickening the membrane to decrease permeability.
The hopanoid diplopterol orders membranes by interacting with lipid A, a common membrane lipid in bacteria, in ways similar to how cholesterol and sphingolipids interact in eukaryotic plasma membranes. Diplopterol and cholesterol were demonstrated to promote condensation and inhibit gel-phase formation in both sphingomyelin monolayers and monolayers of glycan-modified lipid A. Furthermore, both diplopterol and cholesterol could rescue pH-dependent phase transitions in glycan-modified lipid A monolayers. The role of hopanoids in membrane-mediated acid tolerance is further supported by observations of acid-inhibited growth and morphological abnormalities of the plasma membrane in hopanoid-deficient bacteria with mutant squalene-hopene cyclases.
Hopanoids are produced in several nitrogen-fixing bacteria. In the actinomycete Frankia, hopanoids in the membranes of vesicles specialized for nitrogen fixation likely restrict the entry of oxygen by making the lipid bilayer more tight and compact. In Bradyrhizobium, hopanoids chemically bonded to lipid A increase membrane stability and rigidity, enhancing stress tolerance and intracellular survival in Aeschynomene legumes. In the cyanobacterium Nostoc punctiforme, large quantities of 2-methylhopanoids localize to the outer membranes of survival structures called akinetes. In another example of stress tolerance, hopanoids in the aerial hyphae of the prokaryotic soil bacteria Streptomyces are thought to minimize water loss across the membrane to the air.

Biosynthesis

Squalene synthesis

Since hopanoids are a C₃₀ terpenoid, biosynthesis begins with isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which are combined to form longer chain isoprenoids. Synthesis of these smaller precursors proceeds either via the mevalonate pathway or the methylerythritol-4-phosphate pathway depending on the bacterial species, although the latter tends to be more common. DMAP condenses with one molecule of IPP to geranyl pyrophosphate, which in turn condenses with another IPP to generate farnesyl pyrophosphate. Squalene synthase, coded for by the gene sqs, then catalyzes the condensation of two FPP molecules to presqualene pyrophosphate before oxidizing NADPH to release squalene. However, some hopanoid-producing bacteria lack squalene synthase and instead use the three enzymes HpnC, HpnD and HpnE, which are encoded in the hpn operon with many other hopanoid biosynthesis genes. In this alternative yet seemingly more widespread squalene synthesis pathway, HpnD releases pyrophosphate as it condenses two molecules of FPP to PSPP, which HpnC converts to hydroxysqualene, consuming a water molecule and releasing another pyrophosphate. Then, hydroxysqualene is reduced to squalene in a dehydration reaction mediated by the FAD-dependent enzyme HpnE.

Cyclization

Next, a squalene-hopene cyclase catalyzes an elaborate cyclization reaction, engaging squalene in an energetically favorable all-chair conformation before creating five cycles, six covalent bonds, and nine chiral centers on the molecule in a single step. This enzyme, coded for by the gene shc, has a double ⍺-barrel fold characteristic of terpenoid biosynthesis and is present in the cell as a monotopic homodimer, meaning pairs of the cyclase are embedded in but do not span the plasma membrane. In vitro'', this enzyme exhibits promiscuous substrate specificity, also cyclizing 2,3-oxidosqualene.
Aromatic residues in the active site form several unfavorable carbocations on the substrate which are quenched by a rapid polycyclization. In the last substep of the cyclization reaction, after electrons comprising the terminal alkene bond on the squalene have attacked the hopenyl carbocation to close the E ring, the C₂₂ carbocation may be quenched by mechanisms that lead to different hopanoid products. Nucleophilic attack of water will yield diplopterol, while deprotonation at an adjacent carbon will form one of several hopene isomers, often diploptene.

Functionalization

After cyclization, hopanoids are frequently modified by hopanoid biosynthesis enzymes encoded by genes in the same operon as shc, hpn. For instance, the radical SAM protein HpnH adds an adenosine group to diploptene, forming the extended C₃₅ hopanoid adenosylhopane, which can then be further functionalized by other hpn gene products. HpnG catalyzes the removal of adenine from adenosylhopane to make ribosyl hopane, which reacts to form bacteriohopanetetrol in a reaction mediated by an unknown enzyme. Additional modifications may occurs as HpnO aminates the terminal hydroxyl on BHT, producing amino bacteriohopanetriol, or as the glycosyltransferase HpnI converts BHT to N-acetylglucosaminyl-BHT. In sequence, the hopanoid biosynthesis associated protein HpnK mediates deacetylation to glucosaminyl-BHT, from which radical SAM protein HpnJ generates a cyclitol ether.
Importantly, C₃₀ and C₃₅ hopanoids alike may be methylated at C₂ and C₃ positions by the radical SAM methyltransferases HpnP and HpnR, respectively. These two methylations are particularly geostable compared to side-chain modifications and have entertained geobiologists for decades.
In a biosynthetic pathway exclusive to some bacteria, the enzyme tetrahymanol synthase catalyzes the conversion of the hopanoid diploptene to the pentacyclic triterpenoid tetrahymanol. In eukaryotes like Tetrahymena, tetrahymanol is instead synthesized directly from squalene by a cyclase with no homology to the bacterial tetrahymanol synthase.

In paleobiology

Hopanoids have been estimated to be the most abundant natural products on Earth, remaining in the organic fraction of all sediments, independent of age, origin or nature. The total amount in the Earth was estimated as 10 x 10¹⁸ gram in 1992. Biomolecules like DNA and proteins are degraded during diagenesis, but polycyclic lipids persist in the environment over geologic timescales due to their fused, stable structures. Although hopanoids and sterols are reduced to hopanes and steranes during deposition, these diagenetic products can still be useful biomarkers, or molecular fossils, for studying the coevolution of early life and Earth.
Currently, the oldest detected undisputed triterpenoid fossils are Mesoproterozoic okenanes, steranes, and methylhopanes from a 1.64 Ga old basin in Australia. However, molecular clock analyses estimate that the earliest sterols were likely produced around 2.3 Ga ago, around the same time as the Great Oxidation Event, with hopanoid synthesis arising even earlier.
For several reasons, hopanoids and squalene-hopene cyclases have been hypothesized to be more ancient than sterols and oxidosqualene cyclases. First, diplopterol is synthesized when water quenches the C₂₂ carbocation formed during polycyclization. This indicates that hopanoids can be made without molecular oxygen and could have served as a sterol surrogate before the atmosphere accumulated oxygen, which reacts with squalene in a reaction catalyzed by squalene monooxygenase during sterol biosynthesis. Furthermore, squalene binds to squalene-hopene cyclases in a low-energy, all-chair conformation while oxidosqualene is cyclized in a more strained, chair-boat-chair-boat conformation. Squalene-hopene cyclases also display more substrate promiscuity in that they cyclize oxidosqualene in vitro, causing some scientists to hypothesize that they are evolutionary predecessors to oxidosqualene cyclases. Other scientists have proposed that squalene-hopene and oxidosqualene cyclases diverged from a common ancestor, a putative bacterial cyclase that would have made a tricyclic malabaricanoid or tetracyclic dammaranoid product.