_________________________________________________________________________
MPS 587
MBioS571
Advanced Plant Biochemistry
PLANT TERPENOIDS: METABOLISM AND FUNCTION
Mark Lange
Institute of Biological Chemistry
Washington State University
Lecture 1
Outline
1.
1.1
1.2
Historical Overview
Terpenoid Hall of Fame
Paleogeochemistry
2.
2.1
2.2
Biosynthetic Principles
Classification
Retrobiosynthesis - Examples
3.
3.1
3.2
3.3
Metabolic Pathways
The 4 Stages of Isoprenoid Metabolism
The 2 Pathways for IPP/DMAPP Biosynthesis
Pathway Crosstalk
4.
Selected References and Links
_________________________________________________________________________
1.
Historical Overview
1.1
Terpenoid Hall of Fame
1870s: First members were isolated from turpentine (German:
“Terpentin”)
1887: Head to tail condensations of isoprene units (isoprene rule)
"head"
"tail"
August Kékulé (1829 – 1896)
Otto Wallach (1847 – 1931)
Nobel Prize 1910
Isoprene
1910: First structural elucidation of a sesquiterpene (betasantalene)
1910s: Role of pigments in photosynthesis
BM Lange
Adolf von Baeyer (1835 – 1917)
Nobel Prize 1905
Friedrich Wilhelm Semmler (1860 – 1931)
Richard Willstätter (1872 – 1942)
Nobel Prize 1915
Page 1
1.1
Terpenoid Hall of Fame (continued)
1910s: Constitution of bile acids
1910s: Constitution of sterols and their connection with vitamins
1922: Biogenetic isoprene rule (emphasis on biochemical origin,
not chemical structure)
1920s and 1930s: Structural Elucidation of sexual hormones
1920s and 1930s: carotenoids as (pro-) vitamins
1930s: Synthesis of carotenoids
1930s: Role of (pro)vitamin A in vision
1950s: Terpenoids are derived from “active isoprene” via
mevalonic acid
CH2O P P
CH2O P P
Isopentenyl diphosphate
Feodor Lynen (1911 – 1979)
Konrad Bloch (1912 – 2000)
Nobel Prize 1964
Dimethylallyl diphosphate
1950s to 1960s: Synthetic progesterone as oral contraceptive
1951: First total synthesis of non-aromatic steroids
1950s and 1960s: Gibberellins as plant hormones
1950s and 1960s: Cytokinins as plant hormones
1963: Abscisic acid as plant hormone
1970s: Regulation of cholesterol biosynthesis
1970s and 1980s: Elongation and cyclization mechanisms; headto-head condensations
1980s: 3D structure of photosynthetic reaction center
1980s: Brassinosteroids as plant hormones
1990s: Mevalonate-independent (nonMVA) pathway discovered
1994: Total synthesis of taxol
1997: Cystal structure of sesquiterpene synthases
1997: Cystal structure of squalene synthase
1990s and 2000s: Biosynthesis of taxol; cyclization mechanisms
1990s and 2000s: nonMVA pathway elucidated by genomics
BM Lange
Heinrich Wieland (1877 – 1957)
Nobel Prize 1927)
Adolf Windaus (1876 – 1959)
Nobel Prize 1928
Leopold Ružička (1887 – 1976)
Nobel Prize 1939
Adolf Butendandt (1903 - 1995)
Nobel Prize 1939
Paul Karrer (1889 – 1971)
Nobel Prize 1937
Richard Kuhn (1900 – 1967)
Nobel Prize 1938
George Wald (1906-1997)
Nobel Prize 1967
Carl Djerassi (1923*)
Robert Robinson (1885 – 1975)
Nobel Prize 1947
Robert Woodward (1917 – 1979)
Nobel Prize 1965
John Cornforth (1917*)
Nobel Prize 1975
Bernard Phinney
Folke Skoog (1908 – 2001)
Carlos Miller
David Letham
Frederick Addiott
Philip Wareing
Van Steveninck
Michael Brown (1941*)
Joseph Goldstein (1940*)
Nobel Prize 1985
Dale Poulter
Johann Deisenhofer (1943*)
Robert Huber (1937*)
Hartmut Michel (1948*)
Nobel Prize 1988
J.W. Mitchell
T. Yokota
Michel Rohmer
Duilio Arigoni
Robert Holton
K.C. Nicolau
David Cane
David Christianson
Joseph Noel
Joseph Chappell
Georg Schulz
Rodney Croteau
A global race with many players
Page 2
1.1
Paleogeochemistry
Age
[Myr]
0
Cenozoic
Mesozoic
250
Paleozoic
Oleanane
(Angiosperms)
4-Methylstearanes
(Dinoflagellates)
Tricyclic diterpenoids
(Gymnosperms)
500
Aryl terpenoids
(Phototrophic bacteria)
750
Proterozoic 3
Gammacerane
(Protozoa)
1000
1250 Proterozoic 2
C26 Steranes
(Eukaryotes)
1500
Dinorphane
(Prokaryotes)
C26 – C30 Steranes
(Eukaryotes)
C27 – C35 Hopanes
(Eukaryotes)
1750
Proterozoic 1
Biphytanes
(Archaebacteria)
2000
2.
Biosynthetic Principles
Formation of isoprenoids by condensation of C5 units:
“Head” to “tail”
OH
O
H
Monoterpenes
(Menthone)
Sesquiterpenes
(Cedrol)
“Head” to “head”
Triterpenes (squalene as intermediate)
BM Lange
Tetraterpenes (β-Carotene)
Page 3
“Head” to “middle”
Monoterpene
(Pyrethrin)
2.1
Classification
It was originally believed that the smallest naturally occuring family of isoprenoids contained 10 carbons (2 x
C5), which was thus termed “monoterpenes”
C5
Hemiterpenes
(1 x C5)
C25 Sesterterpenes
(5 x C5)
C10 Monoterpenes
(2 x C5)
C30 Triterpenes
(6 x C5)
C15 Sesquiterpenes
(3 x C5)
C40 Tetraterpenes
(8 x C5)
C20 Diterpenes
(4 x C5)
C>40 Polyterpenes
(n x C5; n > 8)
Hemiterpenes
Isoprene
- each year hundreds of millions of metric tons
are emitted into the global atmosphere by a
variety of C3 plants
- function still a matter of debate
Monoterpenes
O
(-)-Limonene
(citrus aroma)
β-Pinene
part of conifer
essential oils
(-)-Menthone
main component
of peppermint
essential oil
Sesquiterpenes
OH
OH
H
(E)-β-Farnesene
(aphid alarm pheromone)
(+)-Cedrol
part of cedar
essential oil
HO
CH3
CH3
Capsidiol
phytoalexin of pepper
and tobacco
Diterpenes
AcO
O
OH
H
H
COOH
RO
CHO
GA12 aldehyde
precursor of plant
hormone
BM Lange
COOH
Abietic acid
part of oeloresin
secreted by Grand fir
O
AcO
OH OBz
R = N-benzoyl-3-phenylisoserine
Taxol TM; Paclitaxel
(antitumor activity)
Page 4
Triterpenes
OH
OH
HO
HO
HO
H
O
β-Sitosterol
O
Brassinolide
(plant hormone)
Tetraterpenes
OH
O
O
HO
Violaxanthin (component of xanthophyll cycle)
β-Carotene (accessory pigment)
Polyterpenes
O
O
H3 CO
H
H
n
HO
9
O
H3 CO
H
3
O
O
Ubiquinone
n = 9, Q-9; n = 10, Q-10
(mitochondrial electron transport)
Plastoquinone-9
(photosynthetic electron transport)
α-Tocopherol; Vitamin E
(plastidial membrane antioxidant)
HO
Phytol
(forms phytyl side chain of chlorophyll)
Meroterpenoids
OH
HN
N
N
N
O
N
Tetrahydrocannabinol; THC
(active principle of cannabis)
trans-Zeatin
(plant hormone)
2.1
Retrobiosynthesis
OH
O
H
BM Lange
Page 5
Practice examples:
H3CCOO
O
OH
O
O
O
OH
HO
O
OH
OOCCH3
O
O
H
H
COOH
CHO
Try it again:
H3CCOO
O
OH
O
O
O
HO
OH
O
OH
OOCCH3
O
O
H
H
COOH
BM Lange
CHO
Page 6
3.
Metabolic Pathways
3.1
The 4 Stages of Isoprenoid Metabolism
Acetate/mevalonate
(MVA) pathway
Deoxyxylulose-5phosphate (DXP)
pathway
Pyruvate
+
D-GAP
Acetyl-CoA
1-Deoxy-D-xylulose5-phosphate
1
Mevalonate
CH2O P P
Hemiterpenes
(C5)
CH2O P P
DMAPP
IPP
Cytokinins
(C5-prenylated)
1x
2x
2
GPP
3x
Monoterpenes
(C10)
FPP
Sesquiterpenes
(C15)
Triterpenes
(e.g., sterols; C30)
nx
GGPP
Brassinosteroids
Diterpenes
(e.g., gibberellins; C20)
Phytol side chain
(e.g., tocopherols, chlorophylls, phylloquinone; C20)
Tetraterpenes
Abscisic acid
(e.g., carotenoids; C40)
PolyPP
Polyterpenes
(e.g., dolichols, rubber, side chains of plastoquinone and ubiquinone)
3
4
1
Synthesis of isopentenyl diphosphate (IPP) via either the MVA pathway or the recently
discovered DXP pathway
2
Isomerization of IPP to DMAPP (IPP isomerase), and subsequent condensation reactions
of DMAPP with IPP to yield GPP, FPP and GGPP (prenyltransferases)
3
Cyclization of allylic diphosphates to basic isoprenoid skeleta (terpene synthases)
4
Secondary modification reactions (e.g. hydroxylations, oxidations, reductions, dehydrations,
isomerizations.)
Abbreviations: DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GAP, glyceraldehyde-3-phosphate;
GPP, geranyl diphosphate; GGPP, geranyl geranyl diphosphate; IPP, isopentenyl diphosphate;
MVA, mevalonate
BM Lange
Page 7
3.2
The 2 Pathways for IPP/DMAPP Biosynthesis
In plants, two spatially separated pathways operate for IPP and DMAPP biosynthesis:
Acetate/mevalonate (MVA) pathway (localized to cytosol)
Ac-CoA
CoA-SH
Ac-CoA
O
O
S-CoA
AcetylCoA
CoA-SH
OH O
O
S-CoA 3-Hydroxy-3-methylglutarylCoA synthase; HMGS
Acetoacetyl(Montamat et al. (1995) Gene
CoA
167:, 197-201)
Acetoacetyl-CoA thiolase;
AACT
(Vollack, Bach (1996) Plant
Physiol 111:, 1097-1107)
HOOC
S-CoA
HMG-CoA
3-Hydroxy-3-methylglutarylCoA reductase; HMGR
(Learned, Fink (1989) Proc
Natl. Acad Sci USA 86:, 27792783)
NADPH
NADP+
ATP
ADP
ADP
OH
HOOC
OH
Mevalonate kinase; MK HOOC
OH
(Riou et al. (1994) Gene
Mevalonate
148:, 293-297)
OH
O P P
Mevalonatediphosphate
Phosphomevalonate
kinase
(plant gene not yet
characterized)
HOOC
ATP
O P
Mevalonatephosphate
ATP
Mevalonate diphosphate
decarboxylase; MVD
(Cordier et al. (1994) Plant Mol
Biol 39:, 953-967)
ADP
CO2
H2O
CH2O P P
CH2O P P
Isopentenyl
diphosphate
In general:
Dimethylallyl
diphosphate
Supply of precursors for the majority of isoprenoids derived from FPP and squalene
Synthesis of intermediates in triterpene, ubiquinone and polyterpene biosynthesis (exception:
plastoquinone)
Mevalonate-independent pathway of IPP/DMAPP biosynthesis (localized to plastids)
H
CO2
O
COOH
+
O
NADP+
OH
CH2 O P
OH
D-Glyceraldehyde-
Pyruvate
NADPH
OH
CH2O P
Deoxyxylulose-5-phosphate
synthase; DXPS
(Lange et al. (1998) Proc Natl
Acad Sci USA 95: 2100-2104)
O
H+
3-phosphate
CH2 O P
Deoxyxylulose-5-phosphate
reductoisomerase; DXR
(Lange et al. (1998) Arch
Biochem Biophys 365: 170-174)
OH
1-DeoxyD-xylulose5-phosphate
OH
OH
2-C-MethylD-erythritol4-phosphate
C
CTP
2-C-Methyl-D-erythritol 4-phosphate
cytidyltransferase; MCT
(Rohdich et al. (2000) Proc Natl Acad
Sci USA 97: 6451-6456)
T
PPi
NH2
OH OH
O P O
O
P
O
O
OH
N
2-C-Methyl-Derythritol2,4-cyclodiphosphate
2 [4Fe-4S]2+
H2O
2-C-Methyl-D-erythritol
2,4-cyclodiphosphate
synthase; MECPS
(plant gene not yet
characterized)
(E)-4-Hydroxy-3-methylbut2-enyl diphosphate
synthase; HDS
(Querol et al. (2002) FEBS
Lett 514: 343-346
2 [4Fe-4S]+
O P P
OH
In general:
BM Lange
ATP
N
O P
OH
2 [4Fe-4S]+
NH2
ADP
OH
CH2O P P O
OH
O
N
O
OH
OH OH
4-(Cytidine 5'-diphospho)2-C-methyl-D-erythritol
kinase; CMK
(Rohdich et al. (2000) Proc Natl
Acad Sci USA 97: 8251-8256)
CH2O P P O
OH
O
N
O
OH
OH OH
CH2O P P
2 [4Fe-4S]2+
(E)-4-Hydroxy-3-methylbut2-enyl diphosphate
reductase; HDR
(Hsieh and Goodman (2005)
Plant Physiol 138: 641-653
Isopentenyl
diphosphate
CH2O P P
Dimethylallyl
diphosphate
Supply of precursors for the majority of isoprenoids derived from GPP and GGPP
Synthesis of intermediates for the biosynthesis of monoterpenes, diterpenes, tetraterpenes,
and the side chains of chlorophylls, tocopherols, phylloquinone and plastoquinone)
Page 8
3.3
Pathway Crosstalk
Glyceraldehyde
3-phosphate
Pyruvate
1-DeoxyD-xylulose
5-phosphate
Thiamin
and
Pyridoxol
2-C-Methyl-D-erythritol
4-phosphate
L
e
u
c
o
p
l
a
s
t
4-(Cytidine 5’-diphospho)2-C-methyl-D-erythritol
2-Phospho-4-(cytidine 5’diphospho)-2-C-methylD-erythritol
2-C-Methyl-D-erythritol
2,4-cyclodiphosphate
Hydroxymethylbutenyl
4-diphosphate
Dimethylallyl
diphosphate
Acetoacetyl-CoA
HMG-CoA
MVA
MVA 5-phosphate
MVA 5-diphosphate
?
Isopentenyl
diphosphate
C
y
t
o
s
o
l
Acetyl-CoA
Isopentenyl
diphosphate
Dimethylallyl
diphosphate
Isoprene
Carotenoids
Abscisic
acid
Chlorophylls
Geranyl
diphosphate
Monoterpenes
Geranylgeranyl
diphosphate
Diterpenes
(Gibberellins)
Farnesyl
diphosphate
Sesquiterpenes
24-Methyleneiophenol
Citrostadienol
Campesterol
β-Sitosterol
Phytyl
diphosphate
Tocopherols
and
Phylloquinone
Stigmasterol
Brassinolide
Organism/tissue/genotype Observation
Peppermint secretory cells
Chamomile flowers
Arabidopsis cla1 mutant
Arabidopsis
Solidago canadensis
Arabidopsis
Monoterpenes and sesquiterpenes are derived exclusively from plastidial IPP pools
McCaskill and Croteau (1995) Planta 197: 4956
Isoprene building blocks 1 and 2 of sesquiterpenes bisaboloxide A and chamazulene are Adam et al. (1998) Arch Biochem Biophys 354:
derived from nonMVA pathway; block 3 is derived from MVA and nonMVA pathways 181-187
Exongenous MVA partially restores chloroplast development and pigment biosynthesis Nagata et al. (2002) Planta 216: 345-350
(usually controlled by the nonMVA pathway) in mutants impaired in the expression of a
nonMVA pathway enzyme
Gibberellin precursor ent-kaurene was synthesized primarioly via nonMVA pathway (> Kasahara et al. (2002) J Biol Chem 277: 4518885 %) with a small contribution of MVA pathway (< 10 %)
45194
Sesquiterpene germacrene D is synthesized predominantly via nonMVA pathway
Steliopoulos et al. (2002) Phytochemistry 60: 1320
In the presence of MVA pathway inhibitor, nonMVA pathway can partially restore
Laule et al. (2003) Proc Natl Acad Sci USA
decreased pools of cytosolic IPP; MVA pathway can not restore depleted pools of
100: 6866-6871
plastidial IPP caused by addition of nonMVA pathway inhibitor
Tobacco BY-2 suspension
cells
In the presence of an MVA pathway inhibitor, sterols (which are usually derived from Hemmerlin et al. (2003) J Biol Chem 278:
the MVA pathway) are synthesized via the nonMVA pathway; in the presence of a
26666-26676
nonMVA pathway inhibitor, plastoquinone (which is usually derived from the nonMVA
pathway) is labeled by exogenously proferred MVA (no quantitation performed)
Spinach, kale, Indian
mustard
Partial purification of a unidirectional (plastid to cytosol) transporter for isoprenoid
intermediates
Bick and Lange (2003) Arch Biochem Biophys
415: 146-154
Arabidopsis phyB mutant
phytochrome B mutant plants are resistant to a nonMVA pathway inhibitor; this is
interpreted by the authors as an increased ability to import isoprenoid precursors into
plastids (no further experimental evidence provided)
Rodriguez-Concepcion et al. (2004) Plant Cell
16: 144-156
Arabidopsis
Prenyl group of trans-zeatin (most likely a plastidial cytokinin) is produced primarily
Kasahara et al. (2004) J Biol Chem 279: 1404914054
via nonMVA pathway (> 80 %); in contrast, the majority of DMAPP used for the
synthesis of cis-zeatin (most likely used for cytosolic tRNA prenylation) stems from the
MVA pathway (roughly 60 % MVA, 40 % nonMVA)
Snapdragon petals
Monoterpenes and sesquiterpenes are synthesized exclusively via the nonMVA pathway Dudareva et al. (2005) Proc Natl Acad Sci USA
102: 933-938
Monoterpenes are almost exclusively synthesized via the nonMVA pathway, whereas Hampel et al. (2005) J Agric Food Chem 53:
sesquiterpenes are generated via the MVA and nonMVA pathways at approximately
2652-2657
equal rates
Monoterpenes are synthesized exclusively via the nonMVA pathway, whereas
Hampel et al. (2005) Phytochemistry 66: 305sesquiterpenes are generated by both the MVA and the nonMVA pathways (at a 50:50 311
Vitis vinifera leaves
Carrot roots and leaves
BM Lange
Reference
Page 9
A
120
10
20
30
40
[Ammonium sulfate]
1M
0M
200
150
100
50
80
60
40
20
0
4
3
2
1
0
-5
0
5
10 15
1/[DMAPP] µM 10-3
0
500
1000
1500
2000
2500
[DMAPP] µM
300
200
200
0
10
15
20
F
C-substrate
Km [µmoles]
Rate of uptake
[nmol (hr • mg protein)-1]
C
150
100
50
0
5±3
219 ± 17
108 ± 9
195 ± 12
101 ± 9
13 ± 4
12
8
4
0
-5
0
5 10 15
1/[GPP] µM x10-3
0
500
1000
1500
2000
2500
[GPP] µM
120
Figure 3. (A), Separation of solubilized inner
envelope membrane proteins using hydrophobic
interaction
chromatography;
proteins
were
solubilized with CHAPS and ammonium sulfate added
to a final concentration of 1 M; the soluble protein
fraction was applied to a phenylsepharose column
and proteins were eluted in a gradient of ammonium
sulfate; the protein fractions were reconstituted into
proteoliposomes and [1-14C] IPP transport activity
determined; upper panel: the elution profile to
proteins from the column (monitored at 280 nm)
along with the ammonium sulfate gradient used for
elution (dotted line); lower panel: [1-14C] IPP uptake
activity of the resulting proteoliposomes prepared by
reconstitution of the protein fractions; (B-E) kinetic
analysis of prenyl diphosphate uptake into
proteoliposomes containing reconstituted inner
envelope membrane proteins from spinach
chloroplasts using IPP, DMAPP, GPP, or FPP as
substrates at the concentrations indicated; (F),
substrate specificity of prenyl diphosphate uptake into
reconstituted inner envelope membrane proteins.
Vmax [nmol min-1 mg protein-1]
ND
89
222
124
111
ND
D
100
80
60
40
20
0
30
20
10
0
-5
0
5
10
15
20
1/[FPP] µM x10-3
0
200
400
600
800
1000
1200
[FPP] µM
250
Vmax [nmol min-1 mg protein-1]
MVA
IPP
DMAPP
GPP
FPP
GGPP
16
1/Vmax
[nmol min-1
mg protein-1] x10-3
5
E
200
1/Vmax
[nmol min-1 mg
protein-1] x 10-3
0
1/Vmax
[nmol min-1 mg
protein-1]x10-3
100
Fraction #
14
B
100
0
Vmax [nmol min-1 mg protein-1]
IPP uptake activity
[nmol min-1]
Absorbance 280 [AmU]
250
1/Vmax
[nmol min-1 mg
protein-1 ] x 10-3
0
Vmax [nmol min-1 mg protein-1]
Elution Volume [mL]
-10
150
100
50
30
20
10
0
-20 0
20 40 60
1/[IPP] µM x10-3
0
0
100
200
300
400
500
[IPP] µM
Bick and Lange (2003) Arch Biochem Biophys 415: 146-154
BM Lange
Page
10
4.
Selected References and Links
Croteau R (2000) In Biochemistry and Molecular Biology of Plants (Buchanan, Gruissem, Jones, Eds.) Am
Soc Plant Biol, Waldorf, MD, pp. 1250-1268.
Rodriguez-Concepcion M, Boronat A (2002) Plant Physiol 130:1079-1089.
Fraga BM (2005) Nat Prod Rep 22: 465-486.
Connolly JD, Hill RA (2005) Nat Prod Rep 22: 487-503.
Hanson JR (2005) Nat Prod Rep 22: 594-602.
http://nobelprize.org/nobel/
http://en.wikipedia.org/wiki/Terpenoid
Notes
BM Lange
Page
11
Notes
BM Lange
Page
12