Biosynthesis – Inspiration for Drug Discovery
Biosynthesis of Fatty Acids & Polyketides
Alan C. Spivey
a.c.spivey@imperial.ac.uk
Nov 2008
Format & Scope of Lectures
•
What are fatty acids?
–
–
•
Fatty acid synthesis by Fatty Acid Synthases (FASs)
–
–
•
definitions & variety
Polyketide synthesis by PolyKetide Synthases (PKSs)
–
–
•
eiconasiods: prostaglandins, thromboxanes & leukotrienes
What are polyketides?
–
•
the chemistry involved
the FAS protein complex & the dynamics of the iterative synthesis process
Fatty acid secondary metabolites
–
•
1° metabolites: fatty acids; 2° metabolites: their derivatives
biosynthesis of the building blocks: acetyl CoA & malonyl CoA
the chemistry involved
the PKS protein complexes & the dynamics of the iterative synthesis process
Polyketide secondary metabolites
–
–
–
Type I modular metabolites: macrolides – e.g. erythromycin
Type I iterative metabolites: e.g. mevinolin (=lovastatin®)
Type II iterative metabolites: aromatic compounds and polyphenols: e.g. actinorhodin
Fatty Acid Primary Metabolites
•
Primary metabolites:
–
fully saturated, linear carboxylic acids & derived (poly)unsaturated derivatives:
•
•
•
•
constituents of essential natural waxes, seed oils, glycerides (fats) & phospholipids
structural role – glycerides & phospholipids are essential constituents of cell membranes
energy storage – glycerides (fats) can also be catabolised into acetate → citric acid cycle
biosynthetic precursors – for elaboration to secondary metabolites
OH
OH
OH
glycerol
3x fatty acids
OCOR1
OCOR2
OCOR3
glycerides
SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)]
e.g.
CO2H
8
CO2H
capric acid (C8, n = 3)
1
CO2H
1
16
CO2H
lauric acid (C12, n = 4)
1
12
1
14
CO2H
10
CO2H
caprylic acid (C8, n = 2)
1
1
18
MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g.
myristic acid (C14, n = 5)
palmitic acid (C16, n = 6)
stearic acid (C18, n = 7)
9
9
CO2H
CO2H
1
1
palmitoleic acid (C16, Z -Δ )
oleic acid (C18, Z-Δ )
9
9
16
18
(>80% of fat in olive oil )
POLY-UNSATURATED ACID DERIVATIVES (PUFAs)
e.g.
8
5
CO2H
1
20
11
14
8
5
CO2H
arachidonic acid (AA)
1
(C20, Z -Δ5, Z -Δ8, Z -Δ11, Z -Δ14)
11
14
17
eicosapentaenoic acid (EPA)
(C20, Z-Δ5, Z -Δ8, Z-Δ11, Z -Δ14, Z -Δ17)
20
(in cod liver oil)
Fatty Acids Derivatives – Secondary Metabolites
•
Secondary metabolites
–
further elaborated derivatives of polyunsaturated fatty acids (PUFAs)
•
e.g. polyacetylenes & ‘eicosanoids’ (prostaglandins, thromboxanes & leukotrienes)
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOT OSY NT HESIS 1) 'light reactions': hv -> ATP and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO
HOHO
oligosaccharides
polysaccharides
nucleic acids ( RNA, DNA)
O
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
glycolysis
CO2
SHIKIMAT E METABOLIT ES
cinnamic acid derivatives
aromatic compounds
lignans
PO
CO2
PO
phosphoenol pyruvate
+
HO
HO
O
OH
erythrose-4-phosphate
OH
OH
shikimate
aromatic amino acids
CO2
aliphatic amino acids
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle)
SCoA
CO2
O
malonyl coenzyme A
HO
O
O
acetoacetyl coenzyme A
peptides
proteins
HO
CO2
mevalonate
saturated f atty acids
unsaturated f atty acids
lipids
FAT T Y ACIDS & POLY KET IDES
polyacetylenes
prostaglandins
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
Biosynthesis of Malonyl Coenzyme A
•
Malonyl coenzyme A is the key ‘extender unit’ for the biosynthesis of fatty acids (& polyketides):
–
–
is formed by the carboxylation of acetyl coenzyme A mediated by a biotin-dependent enzyme
this is the first committed step of fatty acid/polyketide biosynthesis (& is a rate controlling step)
Primary metabolism
CO2
Secondary metabolism
GLYCOLYSIS
pyruvate
O
CoASH
CO2
NAD
NADH
oxidative decarboxylation
SCoA
CITRIC ACID CYCLE
acetyl CoA
O
SCoA
O
CoAS
2x CoASH
SCoA
O
acetyl CoA carboxylase
(biotin-dependent)
O
O
acetoacetyl CoA
2x NADPH
CO2
CoASH
2x NADP
HO
CO2
HO
mevalonate (MVA)
SCoA
CO2
O
malonyl CoA
saturated fatty acids
unsaturated fatty acids
lipids
FATTY ACIDS & POLYKETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids
Biosynthesis of Malonyl Coenzyme A
•
Bicarbonate is the source of the CO2:
–
–
–
the bicarbonate is first activated via phosphorylation by ATP
then the phosphorylated bicarbonate carboxylates biotin to give carboxybiotin
then the carboxybiotin carboxylates the enolate of acetyl CoA to give malonyl CoA:
O
bicarbonate
HO
O
Pi
Nu
O
Mg
O
O
ATP
N
NH
H
H
O
NHEnz
S
O
CoAS
enolate of acetyl CoA = Nu
O
Nu
O
ADP
HO
O
P
O
OH
O
HN
H
biotin
carboxylase
NH
H
O
NHEnz
S
O
CoAS
O
O
transcarboxylase
malonyl CoA
biotin = Nu
–
–
the carboxylation of biotin & acetyl CoA are mediated by a single biotin-dependent enzyme (complex)
having both biotin carboxylase and transcarboxylase active sites
NB. coupling to ATP ‘hydrolysis’ provides energy to drive carboxylation processes
Biosynthesis of Fatty Acids – Iterative Oligomerisation
•
fatty acids are biosynthesised from acetyl CoA as a starter unit by iterative ‘head-to-tail’
oligomerisation involving:
–
–
•
condensation with malonyl CoA as an extender unit (with loss of CO2) – a decarboxylative Claisen
condensation
3-step reduction of the resulting ketone → methylene
after n = 2-8 iterations the C8-20 saturated fatty acid is released from the enzyme(s):
Malonyl CoA
O
SEnz
O
O
O
O
O
O
EnzS
EnzS
CoAS
2x NADPH
EnzS
EnzS
2x NADP
+ H2O
EnzS
decarboxylative
Claisen condensation
O
O
EnzS
EnzS
CO2
fatty
acids
O
O
[R]#3
O
EnzS
dCc#1
O
reduction
dCc#3
EnzS
O
[R]#1
dCc#2
[R]#2
EnzS
O
O
The Decarboxylative Claisen Condensation (dCc)
•
in vitro – the classical Claisen condensation:
O
EtO
H
O
OEt
O O
OEt
EtO
O
OEt
O
EtO
+
O
EtO
EtO
•
in vivo - the decarboxylative Claisen condensation catalysed by a ketosynthase (KS)
O
SEnz
O
O
O
O
O
EnzS
EnzS
EnzS
EnzS
O
+
EnzS
CO2
–
–
O
the energy released upon loss of CO2 provides a driving force for the condensation
thioesters are also particularly reactive partners in this type of condensation...
The Claisen Condensation - Why Thioesters?
•
recall the chemistry of coenzyme A (1st lecture) – properties of alkyl thioesters (cf. alkyl esters)
–
–
–
highly electrophilic carbonyl (~ ketone)
high acidity of protons α to the carbonyl of thioesters (cf. ester)
weak C-S bond (cf. C-O bond):
•
due to poor orbital overlap between the p-orbital lone pair on sulfur (nS) [cf. nO] and the carbonyl anti bonding orbital π*C=O;
(i.e. minimal ‘resonance’ nS → π*C=O)
polarised
reacive carbonyl
weak C-S bond
O
O
H
S
R
H
S
less ef f ective
O
O
R
H
cf .
O
H
R
O
2p
3p
pKa = 25
pKa = 20
–
good leaving group ability of RS- (cf. RO-)
•
due to pKa (RSH) ~10 cf. pKa (ROH) ~16
O
Nu:
SR
O Nu
SR
O
cf.
Nu
SR
a good leaving group
R
O
Nu:
OR
O Nu
OR
O
Nu
OR
a poorer leaving group
Ketone → Methylene - Reduction
•
ketone → methylene reduction is achieved via a 3-step process:
1. NADPH-mediated ketone → alcohol reduction catalysed by a keto reductase (KR)
2. syn-eliminataion of water catalysed by a dehydratase (DH)
3. NADPH-mediated hydrogenation of the double bond catalysed by an enoyl reductase (ER)
O
ketone -> alcohol
reduction
O
O
EnzS
OH
synelimination
EnzS
alkene
hydrogenation
O
O
EnzS
EnzS
HS
O
H O
HS
NADPH
•
N
NADP
NH2
NH2
N
NADPH
EnzS
H
O
HR O
H
NH2
NH2
N
H2O
O
N
NADP
all steps are generally stereospecific but stereospecificity varies from organism to organism
–
indicated specificities are for human FAS
Biosynthesis of Fatty Acids – Overview of FAS
•
The in vivo process by which all this takes place involves a ‘molecular machine’ - Fatty Acid
Synthase (FAS)
–
–
–
Type I FAS: single multifunctional protein complex (e.g. in mammals incl. humans)
Type II FAS: set of discrete, dissociable single-function proteins (e.g. in bacteria)
All FASs comprise 8 components (ACP & 7× catalytic activities): ACP, KS, AT, MT, KR, DH, ER & [TE] :
O
O
O
O
S
AT
CoAS
O
O
MT
SH
KS
S
S
O
decarboxylative
Claisen
condensation
SH
S
reduction
SH
1) KR
2) DH
3) ER
n = 2-8
cycles
ACP
n
O
S
SH
O
S
SH
SH
S
translocation
O
TE
OH
O
KS ACP
KS ACP
KS ACP
CoAS
n
SH
O
O
O
KS
ACP
KS ACP
KS ACP
KS = keto synthase (also known as CE = condensing enzyme); AT = acetyl transferase; MT = malonyl transferase;
KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
O
The Acyl Carrier Protein (ACP)
•
•
the Acyl Carrier Protein (ACP) is the key protein that allows the growing oligomer to access the
appropriate active sites
The ACP is first primed by the post-translational modification of one of its serine hydroxyl groups:
–
the introduction of a phosphopantetheine ‘swinging-arm’ by reaction with acetyl coenzyme A:
NH2
N
O
HS
N
H
O
N
H HO
H
ACP OH
O
N
H
O
HO P O
O O
pantetheinate chain
HS
N
O P O P O
O O O O
N
N
coenzyme A (CoASH)
OH
phpsphopantetheinyl transferase
(ACP synthase)
O Me Me
N
H
O
OH
-
P
O ACP
primed acyl carrier protein
O O
phosphopantetheine chain
~ 20Å
–
–
this swinging-arm provides flexibility for module-module acyl transfer & provides binding energy for catalysis
the ACP is inactive prior to priming
Human Fatty Acid Synthase (FAS)
•
the first three-dimensional structure of human fatty acid synthase (272 kDa) at 4.5 Å resolution by Xray crystallography:
–
Maier, Jenni & Ban Science 2006, 311, 1258 (DOI) ; also Fungal FAS @ 3.1 Å resolution see: Jenni et al.
Science 2007, 316, 254 & 288
Structural overview. (A) Front view: FAS consists of a lower part comprising the KS (lower body) and MAT domains (legs) connected at
the waist with an upper part formed by the DH, ER (upper body), and KR domains (arms). (B) Top view of FAS with the ER and KR
domains resting on the DH domains. (C) Bottom view showing the arrangement of the KS and MAT domains and the continuous
electron density between the KS and MAT domains
FATTY ACID BIOSYNTHESIS (type II FAS)
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
SH
NB. the following sequence of slides have been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
O
SH
Me
S
Co
Acetyl-CoA
• AT1 loads acetyl group onto KS1
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
SH
KR1
DH1
ER1
ACP2
MT2
S
Me
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
KR1
DH1
ER1
ACP2
S
Me
SH
O
-O
O
S
MT2
Co
Malonyl-CoA
• AT1 loads malonyl group onto ACP1
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
S
O
DH1
ER1
ACP2
MT2
S
Me
SH
O
O
KR1
-
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
O
Pantetheine
KR1
DH1
ER1
ACP2
S
Me
CO2
S
O
O
O
MT2
-
• KS1 catalyzes Claisen condensation
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
MT2
SH
Pantetheine
S
O
O
Me
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
S
DH1
ER1
ACP2
MT2
Me
O
O
SH
• KR1 catalyzes reduction of ketone
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
S
DH1
ER1
ACP2
MT2
Me
O
OH
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
S
ER1
ACP2
MT2
Me
O
OH
SH
• DH1 catalyzes dehydration of alcohol
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
S
ER1
ACP2
MT2
Me
O
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
S
ACP2
MT2
Me
O
SH
• ER1 catalyzes reduction of alkene
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
S
ACP2
MT2
Me
O
SH
FATTY ACID BIOSYNTHESIS
ACP1
AT1
KS1
Cys
Pantetheine
KR1
DH1
ER1
ACP2
MT2
KS2
Cys
SH
S
O
Me
SH
• KS2 catalyzes translocation to module 2
SH
KR
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
S
O
Me
SH
KR2
DH2
ER2
TE
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
S
O
Pantetheine
Me
SH
O
-O
O
S
Co
Malonyl-CoA
• MT2 loads malonyl group onto ACP2
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
S
O
Pantetheine
Me
S
O
O
O
-
KR2
DH2
ER2
TE
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
S
O
Pantetheine
Me
S
CO2
O
O
O
-
• KS2 catalyzes Claisen condensation
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
S
O
O
Me
SH
KR2
DH2
ER2
TE
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
TE
Ser
S
Me
O
O
• KR2 catalyzes reduction of ketone
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
TE
Ser
S
Me
O
OH
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
S
ER2
TE
Me
O
OH
• DH2 catalyzes dehydration of alcohol
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
S
ER2
Me
O
TE
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
TE
ER2
S
Me
O
• ER2 catalyzes reduction of alkene
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
SH
KR2
DH2
ER2
S
TE
Me
O
Ser
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
Pantetheine
KR2
DH2
TE
ER2
Ser
SH
S
O
Me
• TE catalyzes transesterification
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
SH
O
O
Pantetheine
SH
Me
Ser
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
SH
O
O
Pantetheine
SH
Me
• TE catalyzes hydrolysis
Ser
H
OH
FATTY ACID BIOSYNTHESIS
H1
ER1
ACP2
MT2
KS2
Cys
KR2
DH2
ER2
TE
Ser
SH
OH
Pantetheine
SH
OH
O
Me
Biosynthesis of Unsaturated Fatty Acids
•
two mechanisms are known for the introduction of double bonds into fatty acids:
–
–
in BACTERIA: anaerobic [O] → monounsaturated FAs (MUFAs)
in MAMMALS, INSECTS & PLANTS: aerobic [O] → MUFAs & polyunsaturated FAs (PUFAs)
Biosynthesis of Prostaglandins & Thromboxanes
•
•
prostaglandins & thromboxanes are derived from further oxidative processing of arachiodonic acid
both are important hormones which control e.g. smooth muscle contractility (blood pressure),
gastric secretion, platelet aggregation & inflammation (<nM activity)
–
various pharmaceuticals including corticosteroids & asprin inhibit biosynthethetic steps in these pathways
Biosynthesis of Leukotrienes
•
leukotrienes are the other main class of 2° metabolites derived from arachiodonic acid
–
they are potent (<nM) inflammatory substances released during allergic reactions
O O
5
H
H
O OH
CO2H
O
CO2H
5-lipoxygenase H
CO2H
H
leukotriene A4 (LTA4)
arachidonic acid
LTC4 synthase
OH
OH
OH
CO2H
fatty acid
C5H11
NRPS
(glutathione)
LTA4 hydrolase
H2N
CO2H
O
CO2H
S
N
H
H
N
O
leukotriene C4 (LTC4)
CO2H
leukotriene B4 (LTB4)
The Polyketide Pathway
•
•
•
Polyketides are also sometimes known as acetogenins
acetyl CoA is also the starting point for the biosynthesis of polyketide secondary metabolites
these metabolites are topologically very different to the fatty acid metabolites but are in fact
synthesised in a very similar fashion. The significant difference is that during the iterative cycle of
chain extension the β-keto group is generally not completely reduced out. This gives rise to huge
structural diversity based around a 1,3-oxygenation pattern & cyclisation to give aromatic compounds
O
O
CoAS
O
O
× (n+1)
O
SEnz
n
n
OH
FATTY ACIDS (FAs)
O
SCoA
O
CoAS
O
O
O
× (n+1)
O
HO
O
n
SEnz
CO2H
OH
POLYKETIDES (PKs)
the polyketide pathway
•
NB. unlike fatty acids. polyketides are NOT biosynthesised by humans – only microorganisms
(bacteria) & fungi
Polyketides
•
the structural variety of polyketide secondary metabolites is very wide:
–
NB. starter units marked in red; extender units in bold black; post oligomerisation appended groups in blue
Me
HO
CO2H
OH
6-methylsalicylic acid
(antibiotic)
OMe O
OH
O
O
CO2H
OH
Me
orsellinic acid
O
CO2H
2
H
O
MeO
OH
citrinin
(kidney toxin
'yellow rice disease')
Cl
Griseofulvin
(treatment for ring
worm infections)
O
OH
O
O
O
H
O
POLYKETIDES
OH
HO
O
OH
OH
OH
O
rapamycin
(immunosuppressant)
NB. a mixed polypropionate/acetate
aflatoxin B1
(mycotoxic carcingen)
O
O
O MeO
OH H
O
H
O
MeO
O
N
CO2H
O
O
O
MeO
O
OH O
actinorhodin
(antibiotic)
OMe
HO
O
OH
6-deoxyerythronolide B
NB. a polypropionate
HO
O
O
OH
O
erythromycin A
(antibiotic)
NMe2
O
O
Me
O
O
Me
O
O
OMe
OH
mevinolin
(=lovastatin®)
(anti-cholesterol)
O
Historical Perspective – ‘The Acetate Hypothesis’
•
1907: James Collie (University of London) effects conversion of dehydroacetic acid to orcinol by
boiling with Ba(OH)2 (while trying to deduce the structure of the former):
H2O
H
OH
O
O
HO
O
HO
OH
OH
O
O
O
dehydroacetic acid
–
•
O
O
2,4,6-triketone
(triketide)
HO
O
O
OH
H2O
O
O
OH
O
orcinol
Collie perceptively postulated the triketone as an intermediate & suggetsed that this might also be an
intermediate in the biosynthesis of orcinol (the ‘polyketide hypothesis’)
1955: Arthur Birch used 14C labelled acetate to show that 6-methylsalicylic acid (ex. Penicillium
patulum) was biosynthesised by head-to-tail oligomerisation of 4 × acetate units and proposed the
following biogenesis – proceeding via a tetraketide intermediate (cf. Collie!):
HO
O
O
4x
14
C labelled
acetate
O
O
OH
O
O
OH
OH
OH
3x H2O
OH
HO
O
O
O
O
OH
2x H2O
OH
O
tetraketide
6-methylsalicilic acid
Biosynthesis of Polyketides – Oligomerisation Steps
•
polyketides are biosynthesised by a process very similar to that for fatty acids
–
the key differences are:
•
•
–
greater variety of starter units, extender units & termination processes
absent or incomplete reduction of the iteratively introduced β-carbonyl groups: ie. each cycle may differ in terms
of KR, DH & ER modules & stereochemistry
this leads to enormous diversity...
Polyketide Diversity
•
O
starter units:
R = Me
R
CoAS
n
O
O
R = Et
i
R = Pr & Pr
CoAS
CoAS
acetyl CoA
•
O
extender units:
CoAS
O
R'
R' = H
R' = Me
R' = Et
butyryl CoA
O
CoAS
O
O
O
O
O
CoAS
O
Me
Et
Me-malonyl CoA (C3)
Et-malonyl CoA (C4)
non-functional or missing KR, DH, ER:
OH
O
no KR
no ER
(S)
(R)
OH
no DH
(Z)
none
missing
(E)
H H
or
or
•
isobutyryl CoA
O
CoAS
malonyl CoA (C2)
•
CoAS
CoAS
propionyl CoA
O
O
O
O
stereochemistry:
1) side chain stereochemistry (determined by KSn)
2) OH stereochemistry (determined by KRn+1)
3) alkene stereochemistry (determined by DHn+1)
•
termination step:
–
depends on nucleophile that releases product at TE stage:
O
EnzS
O
Nu = H2O
HO
O
Nu = OH
intramolecular
O
Nu = CoA
O
CoAS
O
NADH
H
Biosynthesis of Polyketides – Overview of PKS
•
the in vivo process of polyketide synthesis involves PolyKetide Synthases (PKSs):
–
PKSs (except Type II, see later) comprise the same 8 components as FASs. i.e. (ACP & 7× catalytic
activities): ACP, KS, AT, MT, [KR, DH, ER & TE]
Type I PKSs: single (or small set of) multifunctional protein complex(es)
–
•
•
–
modular (microbial) - each ‘step’ has a dedicated catalytic site (→ macrolides)
iterative (fungal) – single set of catalytic sites, each of which may operate in each iteration (cf. FASs) (→
aromatics/polyphenols - generally)
Type II PKSs: single set of discrete, dissociable single-function proteins
•
iterative (microbial) - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols)
O
Type I (modular & iterative):
[Type II see later]
O
O
O
S
SH
AT
SH
MT
KS
S
O
decarboxylative
Claisen
condensation
SH
S
1) ± KR
2) ± DH
3) ± ER
n
cycles
ACP
O
O
n
O
S
SH
O
S
SH
SH
S
translocation
O
TE
OH
O
KS ACP
KS ACP
SH
O
n
S
reduction ?
O
O
O
O
KS ACP
CoAS
O
CoAS
O
O
O
CE
ACP
KS ACP
KS = keto synthase; AT = acetyl transferase; MT = malonyl transferase;
KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
KS ACP
O
POLYKETIDE BIOSYNTHESIS [Type I – (modular)]
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
Pantetheine
SH
KR1
DH1
ER1
ACP2
SH
Pantetheine
SH
NB. the following sequence of slides has also been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
KR1
DH1
ER1
SH
Pantetheine
Pantetheine
SH
SH
O
Me
S
ACP2
Co
Propionyl-CoA
• AT0 loads starting group (propionyl) onto ACP0
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
Pantetheine
S
O
Me
KR1
DH1
ER1
ACP2
SH
Pantetheine
SH
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
Pantetheine
S
O
KR1
DH1
ER1
ACP2
SH
Pantetheine
Me
SH
• KS1 catalyzes translocation to module 1
A
POLYKETIDE BIOSYNTHESIS
ACP0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
ER1
ACP2
Pantetheine
Me
SH
DH1
S
O
Pantetheine
KR1
A
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
SH
O
SH
-O
O
S
Co
Me
Methylmalonyl-CoA
• AT1 loads methylmalonyl group onto ACP1
SH
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
S
O
SH
O
SH
Me
O
-
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
S
O
Pantetheine
Me
CO2
S
O
SH
O
Me
O
-
• KS1 catalyzes Claisen condensation
SH
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
SH
Pantetheine
S
O
SH
SH
* Me
O
Me
Stereocenter
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ER1
ACP2
AT2
Me
*
S
O
O
Me
SH
SH
• KR1 catalyzes reduction of ketone
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
Pantetheine
SH
KR1
DH1
ACP2
AT2
Me
*
S
O
* Me
OH
Stereocenter
SH
ER1
SH
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
SH
Pantetheine
DH1
ER1
ACP2
AT2
Me
*
S
O
* Me
OH
SH
SH
• no DH1 activity
POLYKETIDE BIOSYNTHESIS
P0
AT0
ACP1
AT1
KS1
Cys
KR1
SH
Pantetheine
DH1
ER1
ACP2
AT2
Me
S
*
O
* Me
OH
SH
SH
• no ER1 activity
POLYKETIDE BIOSYNTHESIS
ACP1
AT1
KS1
Cys
KR1
DH1
ER1
ACP2
AT2
KS2
Cys
SH
S
O
Pantetheine
HO
*
*
Me
Me
SH
• KS2 catalyzes translocation to module 2
SH
KR
POLYKETIDE BIOSYNTHESIS
H1
ER1
ACP2
AT2
KS2
Cys
Pantetheine
O
HO
Me
SH
*
DH2
ER2
TE
Ser
S
*
KR2
OH
Me
Biosynthesis of Erythromycin – Type I(modular) PKS
•
6-deoxyerthronolide is a precursor to erythromycin A – bacterial antibiotic (Streptomyces erythreus):
–
–
propionate based heptaketide; 3 multifunctional polypeptides (DEBS1, DEBS2 & DEBS3, all ~350 kDa)
Katz et al. Science 1991, 252, 675 (DOI); Staunton, Leadley et al. Science 1995, 268, 1487 (DOI); Khosla et al. J.
Am. Chem. Soc. 1995, 9105 (DOI); review: Staunton & Weissman Nat. Prod. Rep. 2001, 18, 380 (DOI)
Biosynthesis of Mevinolin – Type I(iterative) PKS
•
mevinolin (=lovastatin®) – cholesterol lowering metabolite of filamentous fungus Aspergillus terreus
–
–
–
–
inhibits HMG-CoA → mevalonate (see next lecture) – rate-limiting step in biosynthesis of cholesterol
acetate based polyketide composed of a diketide and nonaketide linked by an ester
2 × Type I (iterative) PKSs: LNKS and LDKS...both contain MeT (methyl transferase) activities
Hutchinson et al. Science 1999, 284, 1368 (DOI)
3x
O
O
CoAS
3x
O
O
LNKS
O
O
CoAS
2x
O
O
Me
(MeT)
SAM
O
SEnz
Me
O
O
CoAS
OH
O
SEnz
SEnz
Me
OH
DielsAlder
CoAS
1x
CoAS
O
O
nonaketide
3x CO2
3x CO2
2x CO2
OH
O
Me
(MeT)
SAM
O
Me
CoAS
P450
O
OH
OH
O
LDKS
O
OH
SEnz
1x CO2
Me
diketide
O
Me
O
O
mevinolin (=lovastatin®)
O
OH
OH P
450
O
OH
Me
OH
Type II PKSs – Enzyme Clusters (Microbial)
•
Type II PKSs: single set of discrete, dissociable single-function proteins (ACP & 6× catalytic
functions): ACP, KSα, KSβ, [KR, DH, ER, & TE] [NB. NO acetyl or malonyl transferases (AT, MT)]
–
•
•
iterative - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols)
these clusters (generally) use malonate as BOTH starter & extender unit
their ACP proteins are able to load malonate direct from malonyl CoA (no MT required)
the starter malonate is decarboxylated by ‘ketosynthase’ β (KSβ) to give S-acetyl-ACP
the extender malonates undergo decarboxylative Claisen condensations by ketosynthase α (KSα)
–
–
•
these clusters rarely utilise KR, DH or ER activities and produce ‘true’ polyketides:
O
CONH2
Type II (iterative):
O
S
O
SH
O
S
O
O
S
SH
CoAS
O
O
O
O
S
S
O
CoAS
KSα ACP
KSβ ACP
O
KSβ ACP
S
O
KSα ACP
O
n
O
1) ± KR
2) ± DH
3) ± ER
S
SH
O
O
S
SH
SH
S
translocation
O
TE
OH
O
n
cycles
O
n
SH
reduction
(rarely)
SH
O
decarboxylative
Claisen
condensation
KSα ACP
KSα ACP
CONH2
O
O
O
KSα ACP
KSα ACP
KSα ACP
KSβ = 'keto synthase β' (=decarboxylase!); KSα = 'keto synthase α' (=ketosynthase!); KR = keto reductase;
DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
O
Biosynthesis of Actinorhodin – Type II PKS
•
actinorhodin – octaketide bacterial antibiotic (Streptomyces coelicolor)
–
Hopwood Chem. Rev. 1997, 97, 2465 (DOI)
7x
O
O
CoAS
O
CoAS
O
O
PKS
[ACP, KSβ , KSα]
O
O
O
O
O
O
O
O
O
1
octaketide
O
O
16
O
cyclase
O
O
8x CO2
O
O
16
SEnz
O
O
16
O
1
OH
KR
SEnz
O
HO
O
O
SEnz
1
OH
aromatase
OH
OH
O
O
O
OH
16
2
H
OH O
actinorhodin
–
O
octaketide synthesis then cyclisation? (as shown above)
hexaketide synthesis then cyclisation then two further rounds of extension?
indications can sometimes be gleaned from biomimetic syntheses...
16
O
CO2H
OH
O
cyclase
O
1
SEnz
timing of 1st cyclisation and mechanism of control of chain length uncertain
•
•
–
O
O
O
O
1
SEnz
Scope of Structures - Type II PKS
•
microbial polyphenolic metabolites:
pentaketides (5x C2)
OMe O
OH
O
O
OH
octaketides (8x C2)
emodin
eugenone
MeO
HO
OMe
O
hexaketides (6x C2)
nonaketides (9x C2)
OH
O
OH
O
O
O
NH2
tetracycline
plumbagin
Me
OH
heptaketides (7x C2)
H
OH
H
O
MeO
O
O
OH
NMe2
O
OH
decaketides (10x C2)
rabelomycine
rubrofusarin
OH
•
OH
O
many display interesting biological activities...
OH
O
OH
Primary Metabolism - Overview
Primary metabolites
Primary metabolism
Secondary metabolites
CO2 + H2O
PHOT OSY NTHESIS 1) 'light reactions': hv -> AT P and NADPH
2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO
HOHO
oligosaccharides
polysaccharides
nucleic acids ( RNA, DNA)
O
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
glycolysis
CO2
SHIKIMAT E METABOLIT ES
cinnamic acid derivatives
aromatic compounds
lignans, f lavinoids
PO
CO2
PO
phosphoenol pyruvate
+
HO
HO
O
OH
erythrose-4-phosphate
OH
OH
shikimate
aromatic amino acids
CO2
aliphatic amino acids
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle)
SCoA
CO2
O
malonyl coenzyme A
HO
O
O
acetoacetyl coenzyme A
peptides
proteins
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
HO
CO2
mevalonate
saturated f atty acids
unsaturated f atty acids
lipids
FAT T Y ACIDS & POLY KET IDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids