Hopanoids are a diverse subclass of Triterpene 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.
Since their initial discovery in an angiosperm, hopanoids have been found in plasma membranes of bacteria, , , , tropical trees and Fungus.[William W. Christie. "The AOCS Lipid Library. Hopanoids". American Oil Chemists' Society. Archived from the original on 2016-03-05. Retrieved 2015-11-17.]
Biological function
About 10% of sequenced bacterial
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 (e.g., cholesterol), which modulate membrane fluidity and serve other functions in eukaryotes.
The hopanoid diplopterol orders membranes by interacting with lipid A, a common membrane lipid in bacteria, in ways similar to how cholesterol and interact in eukaryotic plasma membranes.
Hopanoids are produced in several nitrogen-fixing bacteria.
Biosynthesis
Squalene synthesis
Since hopanoids are a C
30 terpenoid, biosynthesis begins with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAP), 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 (FPP).
Squalene synthase, coded for by the gene
sqs, then catalyzes the condensation of two FPP molecules to presqualene pyrophosphate (PSPP) 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 (also called
hpnF in some bacteria), has a double ⍺-barrel fold characteristic of terpenoid biosynthesis
and is present in the cell as a monotopic
Protein dimer, meaning pairs of the cyclase are embedded in but do not span the plasma membrane.
, this enzyme exhibits promiscuous substrate specificity, also cyclizing 2,3-oxidosqualene.
Aromatic residues in the active site form several unfavorable on the substrate which are quenched by a rapid polycyclization.
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 C
35 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.
Importantly, C30 and C35 hopanoids alike may be methylated at C2 and C3 positions by the radical SAM methyltransferases HpnP and HpnR, respectively.
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 10
18 gram (10
12 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
during deposition, these diagenetic products can still be useful biomarkers, or
, for studying the coevolution of early life and Earth.
Currently, the oldest detected undisputed triterpenoid fossils are Mesoproterozoic , steranes, and methylhopanes from a 1.64 Ga (billion year) old basin in Australia.
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.
2-methylhopanoids
As a biomarker for cyanobacteria
Proposal
2-methylhopanes, often quantified as the 2-α-methylhopane index, were first proposed as a biomarker for
Photosynthesis by Roger Summons and colleagues following the discovery of the precursor lipids, 2-methylhopanols, in
cultures and mats.
The subsequent discovery of 2-α-methylhopanes supposedly from
photosynthetic cyanobacteria in 2.7 Ga old
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 Anoxic event (OAEs) and the associated 15N isotopic signatures indicative of N2-fixation.
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.
Furthermore, the anoxygenic
phototroph Rhodopseudomonas palustris was found to produce 2-methyl-BHPs only under anoxic conditions.
This latter discovery also lead to the identification of the gene encoding the key methyltransferase HpnP.
was subsequently identified in an acidobacterium and numerous alphaproteobacteria, and phylogenetic analysis of the gene concluded that it originated in the alphaproteobacteria and was acquired by the cyanobacteria and
acidobacteriota via horizontal gene transfer.
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.
Other interpretations
Research demonstrating that the nitrite-oxidizing bacteria (NOB)
Nitrobacter vulgaris increases its production of 2-methylhopanoids 33-fold when supplemented with cobalamin has furthered a non-cyanobacterial explanation for the observed abundance of 2-methylhopanes associated with Cretaceous OAEs. Felix Elling and colleagues propose that overturning circulation brought ammonia- and cobalt-rich deep waters to the surface, promoting aerobic nitrite oxidation and cobalamin synthesis, respectively. This model also addresses the conspicuous lack of 2-methylhopanes associated with Mediterranean
sapropel events and in modern
Black Sea sediments. Because both environments feature much less upwelling, 2-methylhopanoid-producing NOB such as
N. vulgaris are outcompeted by NOB with higher nitrite affinity and
anammox bacteria.
An environmental survey by Jessica Ricci and coauthors using metagenomes and clone libraries found significant correlation between plant-associated microbial communities and hpnP presence, based on which they propose that 2-methylhopanoids are a biomarker for sessile microbial communities high in osmolarity and low in oxygen and fixed nitrogen.
3-methylhopanoids
3-methylhopanoids have historically been associated with aerobic
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.
However, acetic acid bacteria have been known for decades to also produce 2-methylhopanoids.
Additionally, following their identification of
hpnR, the gene responsible for methylating hopanoids at the C
3 position, Paula Welander and Roger Summons identified putative
hpnR homologs in members of alpha-, beta-, and gammaproteobacteria,
actinomycetota,
nitrospirota, candidate phylum NC10, and an
acidobacteriota, as well as in three metagenomes. As such, Welander and Summons conclude that 3-methylhopanoids alone cannot constitute evidence of aerobic methanotrophy.
Applications
Industry
The elegant mechanism behind the protonase activity of squalene-hopene cyclase was appreciated and adapted by chemical engineers at the University of Stuttgart, Germany. Active site engineering resulted in loss of the enzyme's ability to form hopanoids, but enabled Brønsted acid catalysis for the stereoselective cyclization of the
monoterpenoids geraniol, epoxygeraniol, and
citronellal.
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.
Researchers indicate this could be due to direct regulation of transport proteins by hopanoids or indirectly by altering membrane ordering in a way that disrupts the transport system.
