Hopanoids

alt=|thumb|377px|Some representative hopanoids: A. Diploptene, also called 22(29)-hopene B. Diplopterol, also called hopan-22-ol, the hydrated cyclomer of diploptene C. Bacteriohopanetetrol (BHT), a common extended hopanoid D. Hopane, the diagenetic product of A and B that results from reducing conditions during deposition and persists in the rock record. The diagenetic product of C would be an extended C₃₅ hopane.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 (BHPs) 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 (e.g., cholesterol), 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.

Hopanoids are produced in several nitrogen-fixing bacteria. 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 (spore bearing structures) 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 (IPP) and dimethylallyl pyrophosphate (DMAP), which are combined to form longer chain isoprenoids. DMAP condenses with one molecule of IPP to geranyl pyrophosphate, which in turn condenses with another IPP to generate farnesyl pyrophosphate (FPP). 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. This enzyme, coded for by the gene shc (also called hpnF in some bacteria), 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. 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 (BHT) 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. These two methylations are particularly geostable compared to side-chain modifications and have entertained geobiologists for decades.

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 × 10¹⁸ gram (10¹² ton) 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 (billion year) 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. 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.

2-methylhopanoids

alt=Structure of a 2-alpha-methylhopane with the carbons of the base hopane structure numbered according to convention. Five rings of carbon, the first four of which are 6-member while the fifth is 5-member, are arranged such that they each share an edge. In the base structure, the rings are singly methylated at carbon numbers 8, 10, 14, and 18, and doubly methylated at carbon number 4. The twenty-first carbon, located in the fifth ring, is bound to the second carbon in an 8 carbon chain. In 2-alpha-methylhopane, the compound is methylated at carbon number 2. This additional methyl group is indicated in red.|thumb|Structure of a 2-α-methylhopane with the carbons of the base hopane structure numbered according to convention. The methyl group at the C₂ position is indicated in red.

As a biomarker for cyanobacteria

Proposal

2-methylhopanes, often quantified as the 2-α-methylhopane index, were first proposed as a biomarker for oxygenic photosynthesis by Roger Summons and colleagues following the discovery of the precursor lipids, 2-methylhopanols, in cyanobacterial cultures and mats. The subsequent discovery of 2-α-methylhopanes supposedly from photosynthetic cyanobacteria in 2.7 Ga old shales from the Pilbara Craton of Western Australia suggested a 400 Ma (million year) gap between the evolution of oxygenic metabolism and the Great Oxidation Event. However, these findings were later rejected due to potential contamination by modern hydrocarbons.

Putative cyanobacterial presence on the basis of abundant 2-methylhopanes has been used to explain black shale deposition during Aptian and Cenomanian–Turonian Ocean Anoxic Events (OAEs) and the associated ¹⁵N isotopic signatures indicative of N₂-fixation. In contrast, 2-α-methylhopane index values are relatively low across similar Frasnian and Famennian sediments corresponding to the Kellwasser event(s), though higher levels have been reported in later Lower Famennian sections.

Dispute

The status of 2-methylhopanoids as a cyanobacterial biomarker was challenged by a number of microbiological discoveries. Geobacter sulfurreducens was demonstrated to synthesize diverse hopanols, although not 2-methyl-hopanols, when grown under strictly anaerobic conditions. This latter discovery also lead to the identification of the gene encoding the key methyltransferase HpnP.

Among cyanobacteria, hopanoid production is generally limited to terrestrial cyanobacteria. Among marine cyanobacteria, culture experiments in conducted by Helen Talbot and colleagues concluded that only two marine species–Trichodesmium and Crocosphaera–produced bacteriohopanepolyols. A later gene-based search for hpnP in available cyanobacterial genomes and Metagenome Assembled Genomes (MAGs) drew similar conclusions, identifying the gene in ~30% of terrestrial and freshwater species, and only one of the 739 marine cyanobacterial genomes and MAGs. Additionally, Nostoc punctiforme produces the greatest amount of 2-methylhopanoids when differentiated into akinetes. These cold- and desiccation-resistant cell structures are dormant and therefore not photosynthetically active, further challenging the association between 2-methylhopanes and oxygenic photosynthesis.

3-methylhopanoids

3-methylhopanoids have historically been associated with aerobic methanotrophy based on culture experiments and co-occurrence with aerobic methanotrophs in the environment. As such, the presence of 3-methylhopanes, together with ¹³C depletion, are considered markers of ancient aerobic methanotrophy.

Agriculture

The application of hopanoids and hopanoid-producing nitrogen fixers to soil has been proposed and patented as a biofertilizer technique that increases environmental resistance of plant-associated microbial symbionts, including nitrogen-fixing bacteria that are essential for transforming atmospheric nitrogen to soluble forms available to crops.

Medicine

During later studies of interactions between diplopterol and lipid A in Methylobacterium extorquens, multidrug transport was found to be a hopanoid-dependent process. Squalene-hopene cyclase mutants derived from a wild type capable of multidrug efflux, a drug-resistance mechanism mediated by integral transport proteins, lost the ability to perform both multidrug transport and hopanoid synthesis.