Download Biosynthesis of Isoprenoids

Biosynthesis of Isoprenoids
David Wang’s Natural Products Class
Terpene
• Mankind has used terpenes that are extracted from
plants for many different purposes — as fragrances
and flavors, as pharmaceutical agents and as
insecticides.
• Aside from their immense commercial value, terpene
products have important biological functions in plants.
– Plant growth and development (e.g. gibberellin phytohormones)
– Represent important tools in the various interactions of plants
with the environment.
• Volatile and non-volatile terpenes are implicated in the
attraction of both pollinators and predators of
herbivores, in protection against photooxidative stress,
in mediating thermotolerance, and in direct defense
against microbes and insects.
Terpenoids
o Occurrence
n The softwood resin canals are filled with oleoresin.
n Oleoresin: monoterpenoids and especially resin acids
(diterpenoids) are dominant and commercially
important oleoresin constituent.
n The parenchyma resin of both softwoods and
hardwoods contains triterpenoids and steroids, main
occurring as fatty acid esters.
n Some tree produce rubber, gutta percha, and balata,
which polyterpenes.
Classification and Biosynthesis of Terpenoids
n
Terpenoids is formally derived from isoprene units and are
sometimes called isoprenoids.
n
The name terpene was given to hydrocarbons which were
detected in terpentine oil.
n
Terpenes are known as the large group of hydrocarbons
made up of isoprene units (C5H8). Their respective
derivatives with hydroxyl, carbonyl, and carboxyl functions
are not hydrocarbons but strictly speaking terpenoids. →
It has been proposed to call both the terpene
hydrocarbons and their derivatives collectively terpenoids.
Myrcene
Greek Bay (Laurus nobilis)
Hops (Humulus lupulus)
Myrcene is found in the essential oil of bay leaves as
well as hops. It is used as an intermediate in the
manufacture of perfumes.
Geraniol and Linalool
Geraniol
Linalool
Geraniol is isomeric with linalool, constitutes the major
part of the oil of roses and is also found in essential oils
of citronella, lemon grass, and others.
citronella
Menthol
Menthol is a well-known monoterpene which is
found in the essential oil of peppermint and other
members of the mint family.
Sesquiterpenoids
n
More than 10000 sesquiterpenoids have been identified,
representing a wide variety compounds of different
skeletal types from acyclic to tetra cyclic systems.
α-cedrene
α-muurolene
logifloene
δ-cadinene
juniperol
α-cadinol
nootkatin
chanootin
cadinenes
Wormwood
(Artemisia maritima)
santonin
caryophyllene
Dandelion (Taraxacum officinale )
Cloves (Syzygium aromaticum)
tetrahydroridentin B
Classification of Terpenes (Terpenoids)
Classification and Biosynthesis of
Terpenoids
• Isoprene itself had been characterized as a decomposition
product from various natural cyclic hydrocarbons, and was
suggested as the fundamental building block for
isoprenoids.
• Isoprene is produced naturally but is not involved in the
formation of these compounds, and the biochemically
active isoprene units were identified as the DMAPP
(dimethylallyl diphosphate) and IPP (isopentenyl
diphosphate).
Classification and Biosynthesis of Terpenoids
n
Relative few of natural terpenoids conform exactly to the
simply concept of a linear head-to-tail combination of
isoprene units as seen with geraniol, farnesol,
geranylgeraniol.
n
Squalene and phytoene display a tail-to-tail linkage at the
centre of the molecules.
n
Most terpenoids are modified further by cyclization
reactions, but the head-to-tail arrangement of the units
can usually still be recognized such as menthol,
bisabolene, and taxadiene.
Head-to-tail coupling mechanism of terpenoids and steroids
Biosynthesis of Terpenoids and Steroids
Biosynthesis of Terpenoids
n
The prenylation reaction is stereospecific, mediated by a
type of prenyl transferase enzyme, which generates mainly
isoprenoids with trans configuration.
n
In contrast, rubber has an all-cis configuration because its
synthesis is controlled by another type of prenyl transferase
enzyme.
Synthesis of IPP
n
The glandular trichomes and secretory cavities of leaves
and the glandular epidermis of flower petals generate
and store or emit terpenoid essential oils that are
important because they encourage pollination by
insects.
n
The resin ducts and blisters of conifer species the
carbon and energy needed to drive terpenoids
biosynthesis.
Synthesis of IPP
n
A more fundamental, and perhaps universal, feature of the
organization of terpenoids metabolism exists at the
subcellular level.
n
The sesquiterpenes (C15), triterpenes (C30), and
polyterpenes appear to be produced in the cytosolic and
endoplasmic reticulum (ER) compartments, whereas
isoprene, the monoterpenes (C10), diterpenes (C20),
tetraterpenes (C40), and certain prenylated quinones
originate largely, if not exclusively, in the plastids.
Synthesis of IPP
n
The evidence now indicates that the biosynthetic pathways for the
formation of the fundamental precursor IPP differ markedly in these
compartments, with the classical acetate/mevalonate pathway being
active in the cytosol and ER and the glyceraldehyde phosphate/
pyruvate pathway operating in the plastids.
n
Regulation of these dual pathways may be difficult to assess, given
that plastids may supply IPP to the cytosol for use in biosynthesis, and
vice versa.
n
Mitochondria, a third compartment, may generate the ubiquinone
prenyl group by the acetate/mevalonate pathway, although little is
known about the capability of these organelles for terpenoids
biosynthesis.
Head-to-tail coupling mechanism of terpenoids and steroids
萜類化合物之生合成路徑 C30
Organization of terpene biosynthesis in plants
Hydroxymethylglutaryl-CoA reductase, an enzyme in
the acetate/mevalonate pathway, is highly regulated
羥甲基戊二酸輔 A
n
The basic enzymology of IPP
biosynthesis by way of the
acetate/mevalonate pathway
is widely accepted.
n
This cytosolic IPP pathway
involves the two-step
condensation of three
molecules of acetyl-CoA
catalyzed by thiolase and
hydroxymethylglutaryl-CoA
synthase.
Hydroxymethylglutaryl-CoA reductase, an enzyme in
the acetate/mevalonate pathway, is highly regulated
n
The resulting product, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), is
subsequently reduced by HMG-CoA reductase in two coupled
reactions that form mevalonic acid.
n
Two sequential ATP-dependent phosphorylations of mevalonic acid
and a subsequent phosphorylation/elimination-assisted
decarboxylation yield IPP.
HMG-CoA reductase
n
HMG-CoA reductase is one of the most highly regulated
enzymes in animals, being largely responsible for the
control of cholesterol biosynthesis.
n
Accumulated evidence indicates that the plant enzyme,
which is located in the ER membrane, is also highly
regulated.
n
In many cases, small gene families, each containing
multiple members, encode this reductase. These gene
families are expressed in complex patterns, with individual
genes exhibiting constitutive, tissue- or developmentspecific, or hormone-inducible expression.
HMG-CoA reductase
n
Specific HMG-CoA reductase genes can be induced by wounding or
pathogen infection.
n
The activity of HMG-CoA reductase may be subject to post
translational regulation, for example, by a protein kinase cascade that
phosphorylates and thereby inactivates the enzyme.
Allosteric modulation probably also plays a regulatory role. Proteolytic
degradation of HMG-CoA reductase protein and the rate of turnover of
the corresponding mRNA transcripts may also influence enzyme
activity.
n
n
n
Researchers have not arrived at a unified scheme that explains how
the various mechanisms that regulate HMG-CoA reductase facilitate
the production of different terpenoids families.
The precise biochemical controls that influence activity have been
difficult to assess in vitro because the enzyme is associated with the
ER membrane.
In plastids, IPP is synthesized from pyruvate and
glyceraldehyde 3-phosphate
Prenyltransferase and terpene
synthase reactions
異戊烯基轉移酶
n
Prenyltransferase enzymes generate the allylic diphosphate esters
geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and
geranylgeranyl diphosphate (GGPP).
n
Reactions that these compounds undergo (often cyclizations), which
are catalyzed by terpene synthases, yield a wide variety of terpenoid
compounds.
n
Both prenyltransferases and terpene synthases utilize electrophilic
reaction mechanisms involving carbocationic intermediates, a feature
of terpenoid biochemistry.
n
Enzymes in both groups share similar properties and contain
conserved sequence elements, such as an aspartate - rich DDxxD
motif involved in substrate binding, which may participate in initiating
divalent metal ion–dependent ionizations.
Representative terpenoids
biosynthesized by plants.
Monoterpenes, sesquiterpenes,
and diterpenes are derived from
the prenyl diphosphate
substrates geranyl diphosphate
(GPP), farnesyl diphosphate
(FPP), and geranylgeranyl
diphosphate (GGPP),
respectively, and are produced in
both angiosperms and
gymnosperms. (-)-Copalyl
diphosphate and ent-kaurene are
sequential intermediates in the
biosynthesis of gibberellin plant
growth hormones. Taxa-4(5), 11
(12)-diene is the first dedicated
intermediate in the biosynthesis
of Taxol.
Enzyme Limonene Synthase
n
The families of enzymes responsible for the formation of terpenoids
from GPP, FPP, and GGPP are known as monoterpene,
sesquiterpene, and diterpene synthases, respectively.
n
These synthases use the corresponding prenyl diphosphates as
substrates to form the enormous diversity of carbon skeletons
characteristic of terpenoids.
n
Most terpenoids are cyclic, and many contain multiple ring systems,
the basic structures of which are determined by the highly specific
synthases.
n
Terpenoid synthases that produce cyclic products are also referred to
as “cyclases,” although examples of synthases producing acyclic
products are also known.
Enzyme for Limonene Synthase
• A diverse array of monoterpene
synthases has been isolated from
essential oil producing angiosperm
species and resin producing
gymnosperms.
• These enzymes use a common
mechanism in which ionization of
GPP leads initially to the tertiary
allylic isomer linalyl diphosphate
(LPP).
Enzyme for Limonene Synthase
n
This isomerization step is required because
GPP cannot cyclize directly, given the
presence of the trans-double bond.
n
Ionization of the enzyme-bound LPP
intermediate promotes cyclization to a sixmembered ring carbocation (the α-terpinyl
cation), which may undergo additional
electrophilic cyclizations, hydride shifts, or
other rearrangements before the reaction is
terminated by deprotonation of the
carbocation or capture by a nucleophile (e.g.,
water).
Enzyme for Limonene Synthase
n
Variations on this simple mechanistic scheme, involving
subsequent reactions of the α-terpinyl carbocation, are
responsible for the enzymatic formation of most
monoterpene skeletons.
n
The simplest monoterpene synthase reaction is catalyzed by
limonene synthase, a useful model for all terpenoid
cyclizations. The electrophilic mechanism of action used by
limonene synthase can be viewed as an intramolecular
equivalent of the prenyltransferase reaction.
Enzyme for Limonene Synthase
n
Synthases that produce acyclic olefin products (e.g.,
myrcene) and bicyclic products (α- and β-pinene) from
GPP are also known, as are enzymes that transform GPP
to oxygenated derivatives such as 1,8-cineole and bornyl
diphosphate, the precursor of camphor.
n
An interesting feature of the monoterpene synthases is
the ability of these enzymes to produce more than one
product; for example, pinene synthase from several plant
sources produces both α- and β-pinene. The pinenes are
among the most common monoterpenes produced by
plants and are principal components of turpentine of
thepines, spruces, and firs.
Cloning and characterization of
Pinene synthase from Chamaecyparis
formosensis Matsum
氨基酸序列之相似度比較圖 Pa, Picea abies
Ag , Abies grandis
Car, (+)-3-carene synthase
Lim, (-)-limonene synthase
Lin, (-)-linalool synthase
Myr, myrcene synthase
Phe, β-phellandrene synthase
Pin, pinene synthase
628
不同針葉樹mono-TPS基因的親緣關係
52.4%
52%
52.2%
54.3% Pa, Picea abies
Ag , Abies grandis
52.9%
51.7%
53.3%
Car, (+)-3-carene synthase
Lim, (-)-limonene synthase
Lin, (-)-linalool synthase
Myr, myrcene synthase
Phe, β-phellandrene synthase
Pin, pinene synthase
Phylogenetic Tree Showing Cf-Pinene and Other
Reported Coniferous Monoterpene Synthase.
於 E. coli pQE31表現之蛋白質
63 KDa
GC-MS Analysis of Monoterpene Products of Recombinant
Protein Product of Cf-Pin with Geranyl Diphosphate as the
Substrate