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Biosynthesis – Inspiration for Drug Discovery
Biosynthesis of Fatty Acids & Polyketides
Alan C. [email protected]
Nov 2008
Format & Scope of Lectures
• What are fatty acids? – 1° metabolites: fatty acids; 2° metabolites: their derivatives– biosynthesis of the building blocks: acetyl CoA & malonyl CoA
• Fatty acid synthesis by Fatty Acid Synthases (FASs)– the chemistry involved– the FAS protein complex & the dynamics of the iterative synthesis process
• Fatty acid secondary metabolites– eiconasiods: prostaglandins, thromboxanes & leukotrienes
• What are polyketides?– definitions & variety
• Polyketide synthesis by PolyKetide Synthases (PKSs) – 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
CO2H lauric acid (C12, n = 4)
CO2H
palmitoleic acid (C16, Z -Δ9)
112
1
16
9
SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)]e.g.
MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g.
POLY-UNSATURATED ACID DERIVATIVES (PUFAs)e.g.
CO2H arachidonic acid (AA)(C20, Z -Δ5, Z -Δ8, Z -Δ11, Z -Δ14)1
CO2H eicosapentaenoic acid (EPA)(C20, Z-Δ5, Z -Δ8, Z-Δ11, Z -Δ14, Z -Δ17)
(in cod liver oil)
1
2020
CO2H caprylic acid (C8, n = 2)18
CO2H capric acid (C8, n = 3)110
CO2H myristic acid (C14, n = 5)114
CO2H palmitic acid (C16, n = 6)116
CO2H stearic acid (C18, n = 7)118
CO2H
oleic acid (C18, Z-Δ9)(>80% of fat in olive oil )
1
18
9
58
11 14
58
11 14 17
OHOHOH
glycerol
OCOR1
OCOR2
OCOR3
3x fatty acids
glycerides
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
CO2 + H2O
1) 'light reactions': hv -> ATP and NADPH2) 'dark reactions': CO2 -> sugars (Calvin cycle)
OHOHOHOOH
HO
glucose& other 4,5,6 & 7 carbon sugars
Primary metabolism Secondary metabolites
oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)
phosphoenol pyruvate
glycolysis
CO2
HOOH
OHOOH
HO
POCO2
PO
erythrose-4-phosphate
SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans+
shikimate
aromatic amino acids
aliphatic amino acidspeptidesproteinsCO2
Opyruvate
SCoA
Oacetyl coenzyme A
Citr ic acidcycle
(Krebs cycle)
ALKALOIDSpenicillinscephalosporinscyclic peptides
tetrapyrroles (porphyrins)
PHOTOSYNTHESIS
saturated fatty acidsunsaturated f atty acidslipids
FATTY ACIDS & POLYKETIDESpolyacetylenesprostaglandinsaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
SCoA
OCO2
malonyl coenzyme A
CoAS
O
O
HO
HOCO2
mevalonateacetoacetyl coenzyme A
Primary metabolites
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 Secondary metabolism
SCoA
Oacetyl CoA
CO2
SCoAO
CoAS
O
O
saturated fatty acidsunsaturated fatty acidslipids
FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
CO2
NADHNAD
CO2
Opyruvate GLYCOLYSIS
oxidative decarboxylation
CITRIC ACID CYCLE
acetyl CoA carboxylase(biotin-dependent)
SCoAO
HO
HOCO2
mevalonate (MVA)
CoASH
2x NADPH
2x NADP
CoASH
2x CoASH
SCoA
OCO2
malonyl CoA
acetoacetyl CoA
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:
– 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
O
HO O
O
HO OPO
OHO
ATP
ADPO
CoAS O
O
malonyl CoA
Pi
biotin = Nu
NHHN
S
O
HH
O
NHEnz
bicarbonate
CoAS
O
enolate of acetyl CoA = Nu
Nu
NHN
S
O
HH
O
NHEnz
O
OMg
biotincarboxylase transcarboxylase
Nu
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):
O
CoAS
O
EnzS
O
SEnz
O
O O
EnzS
CO2
OEnzS EnzS
O O
EnzS
EnzS
O
reduction
EnzS
O O
EnzS
O
Malonyl CoA
EnzS
O Ofattyacids
decarboxylativeClaisen condensation dCc#1 [R]#1
dCc#2
[R]#2dCc#3
2x NADPH
2x NADP+ H2O
EnzS
O[R]#3
The Decarboxylative Claisen Condensation (dCc)• in vitro – the classical Claisen condensation:
• in vivo - the decarboxylative Claisen condensation catalysed by a ketosynthase (KS)
– 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...
O
OEtH
O
OEt
O
EtO
OEtO O
EtO
O
O
EtO
EtO
+
EtO
O
EnzS
O
SEnz
O
O O
EnzS
CO2
OEnzS
EnzS
O O
EnzS
+
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)
– good leaving group ability of RS- (cf. RO-)• due to pKa (RSH) ~10 cf. pKa (ROH) ~16
O
SR
ORa poorer leaving group
SRa good leaving group
cf.
Nu:
SR
O Nu O
Nu
O
OR
Nu:
OR
O Nu O
Nu
3p
cf .SH R
O
2p
OH R
O
less ef fective
pKa = 25pKa = 20
weak C-S bond
polarisedreacive carbonyl
O
SH R
O
OH R
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)
• all steps are generally stereospecific but stereospecificity varies from organism to organism– indicated specificities are for human FAS
N
HS H
N
EnzS
O OH
HS
syn-elimination
NH2
O
NH2
O
NADP
ketone -> alcoholreduction
H2O
EnzS
O
EnzS
O
EnzS
O
N
H HR
N
NH2
O
NH2
O
NADP
H
alkenehydrogenation
EnzS
O O
NADPH NADPH
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] :
SSO O
O
O
SSH O
O
SSH OSHSO
O
CoAS
O
O
O
CoAS
reduction
KS KSACP ACP
ACPACP KSKS
translocationTE
n
n = 2-8cycles
SHSOO
OH
n
ACP
SHSH
ACP
SHSO
KS ACP
decarboxylativeClaisen
condensationAT
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
MT
1) KR2) DH3) ER
KS
KS
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:
– this swinging-arm provides flexibility for module-module acyl transfer & provides binding energy for catalysis– the ACP is inactive prior to priming
phosphopantetheine chain~ 20Å
OP
O
O-O
MeMeO
OHNH
O
NH
HS
N
NN
N
O
NH2
OHO
OPOPO
OOOO coenzyme A (CoASH)H
O
NH
O
NH
HS
PHO
OO
HO
pantetheinate chain
ACP OH
ACPprimed acyl carrier protein
phpsphopantetheinyl transferase(ACP synthase)
Human Fatty Acid Synthase (FAS)• the first three-dimensional structure of human fatty acid synthase (272 kDa) at 4.5 Å resolution by X-
ray 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
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS (type II FAS)
Pantetheine
Cys
NB. the following sequence of slides have been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
O
Me S Co
• AT1 loads acetyl group onto KS1
Acetyl-CoA
ACP1 AT1 KS1 KR1 DH1 ER1
S
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
OMe
ACP1 AT1 KS1 KR1 DH1 ER1
S
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
OMe
• AT1 loads malonyl group onto ACP1
Malonyl-CoA
O
S Co
O
O-
ACP1 AT1 KS1 KR1 DH1 ER1
S
S
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
OMe
O
O
-O
ACP1 AT1 KS1 KR1 DH1 ER1
S
S
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
OMe
• KS1 catalyzes Claisen condensation
O
O
-O
CO2
ACP1 AT1 KS1 KR1 DH1 ER1
SH
S
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
O
OMe
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
• KR1 catalyzes reduction of ketone
Me
O O
S
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
Me
O OH
S
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
• DH1 catalyzes dehydration of alcohol
Me
O OH
S
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
O
S Me
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
• ER1 catalyzes reduction of alkene
O
S Me
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
O
S Me
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 MT2 KS2
SH
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
Cys
• KS2 catalyzes translocation to module 2
KR
O
Me
S
Cys
H1 ER1 ACP2 MT2 KS2
S
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2
O
Me
TE
SH
CysOH
Ser
H1 ER1 ACP2 MT2 KS2
S
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2
O
Me
TE
SH
• MT2 loads malonyl group onto ACP2
Malonyl-CoA
O
S Co
O
O-
CysOH
Ser
H1 ER1 ACP2 MT2 KS2
S
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2
O
Me
TE
SO
O
-O
CysOH
Ser
H1 ER1 ACP2 MT2 KS2
S
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2
O
Me
TE
S
• KS2 catalyzes Claisen condensation
O
O
-O
CO2
CysOH
Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2
O
Me
TE
SO
CysOH
Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
• KR2 catalyzes reduction of ketone
Me
O O
SCys
OHSer
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
Me
O OH
SCys
OHSer
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
• DH2 catalyzes dehydration of alcohol
Me
O OH
SCysOH
Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TEMe
O
SCysOH
Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
• ER2 catalyzes reduction of alkene
Me
O
SCysOH
Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TEMe
O
S OHCys Ser
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
• TE catalyzes transesterification
OHCys Ser
Me
OS
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
OCys Ser
Me
O
SH
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
• TE catalyzes hydrolysis
OCys Ser
Me
O
SH
OHH
H1 ER1 ACP2 MT2 KS2
SH
FATTY ACID BIOSYNTHESIS
Pantetheine
KR2 DH2 ER2 TE
OHCys Ser
Me
O
SHOH
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
CO2H5-lipoxygenase
O O5
arachidonic acid
H H
CO2HO OH
HH
leukotriene A4 (LTA4)
leukotriene B4 (LTB4)
CO2HOHOH
LTA4 hydrolase
leukotriene C4 (LTC4)
S
LTC4 synthase
CO2HO
HN CO2H
NH
O
CO2H
H2N
O
OHCO2H
C5H11
NRPS(glutathione)
fatty acid
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
• NB. unlike fatty acids. polyketides are NOT biosynthesised by humans – only microorganisms (bacteria) & fungi
O
SCoA
O
SEnz
O
SEnz
O O
O
OH
HO
CO2HOH
n
FATTY ACIDS (FAs)
n
n
POLYKETIDES (PKs)
the polyketide pathway
O
CoAS
O
O
O
CoAS
O
O
× (n+1)
× (n+1)
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
rapamycin(immunosuppressant)
NB. a mixed polypropionate/acetate
6-methylsalicylic acid(antibiotic)
POLYKETIDES
aflatoxin B1(mycotoxic carcingen)
erythromycin A(antibiotic)
mevinolin(=lovastatin®)
(anti-cholesterol)
actinorhodin(antibiotic)
O
O
O
O O
MeO H
H
O
O
O
O
O
OO
NMe2
HO
OH
OMe
HOOH
OH
Me O
O
O O
OH
OHCO2H
HO
CO2HOH
O
O
OHCO2H
MeMe
O
OMe
MeOCl
O
O
O
O OH
orsellinic acid citrinin(kidney toxin
'yellow rice disease')
OH
O
OH
Griseofulvin(treatment for ring worm infections)
6-deoxyerythronolide BNB. a polypropionate
O
O
O
N O
O
O
HO
MeO
MeO
OMe
OH
O
OH H
Me
O
OH
OH
O
O
CO2H2 H
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):
– 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!):
O
O O
OO
H2O
O
HOO
dehydroacetic acidO
OHO
O
O
OH
HO
orcinol
H
H2O
O
OH
OH
O
HOOH
2,4,6-triketone(triketide)
O
OH4x
3x H2O
OOH
O
O O
OOH
HO
O O
OH
HO
O O
OH
OH
OH O2x H2O14C labelled
acetate 6-methylsalicilic acidtetraketide
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 • starter units:
• extender units:
• non-functional or missing KR, DH, ER:
• stereochemistry:
• termination step:– depends on nucleophile that releases product at TE stage:
no KROH
no DHno EROH
or or
O (R) (S) (Z) (E)none
missingH H
O
CoAS
R = MeR = EtR = nPr & iPr
O
CoAS
O
CoAS
O
CoAS R
acetyl CoA propionyl CoA butyryl CoA
O
CoAS
isobutyryl CoA
O
CoAS
O
OR'
R' = HR' = MeR' = Et
malonyl CoA (C2) Me-malonyl CoA (C3) Et-malonyl CoA (C4)
O
CoAS
O
O
O
CoAS
O
OMe
O
CoAS
O
OEt
1) side chain stereochemistry (determined by KSn)2) OH stereochemistry (determined by KRn+1)3) alkene stereochemistry (determined by DHn+1)
Nu = H2OO
EnzS
O
HO
Nu = OHintramolecular
O
ONu = CoA O
CoAS
O
H
NADH
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)
SSO O
O
O
SSH O
O
SSH OSHSO
O
CoAS
O
O
O
CoAS
reduction ?
KS KSACP ACP
ACPACP KSKS
translocationTE
n
ncycles
SHSOO
OH CE
n
ACP
SHSH
ACP
SHSO
KS ACP
decarboxylativeClaisen
condensationAT
KS = keto synthase; AT = acetyl transferase; MT = malonyl transferase;KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
MT
1) ± KR2) ± DH3) ± ER
KSOOO
O
OO
Type I (modular & iterative):[Type II see later]
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 A
POLYKETIDE BIOSYNTHESIS [Type I – (modular)]
Pantetheine
Cys
ACP0 AT0
SH
Pantetheine Pantetheine
NB. the following sequence of slides has also been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 A
Pantetheine
Cys
ACP0 AT0
SH
Pantetheine Pantetheine
OMe S Co
• AT0 loads starting group (propionyl) onto ACP0
Propionyl-CoA
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 A
Pantetheine
Cys
ACP0 AT0
S
Pantetheine Pantetheine
O
Me
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
SH
SH
ACP2 A
Pantetheine
Cys
ACP0 AT0
SPantetheine PantetheineO
Me
• KS1 catalyzes translocation to module 1
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
S
SH
ACP2 A
Pantetheine
Cys
ACP0 AT0
SH
Pantetheine PantetheineO
Me
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
S
SH
ACP2 AT2
SH
Pantetheine
Cys
• AT1 loads methylmalonyl group onto ACP1
Methylmalonyl-CoA
O
S Co
O
O-
P0 AT0
SH
O
Me
Me
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
S
S
ACP2 AT2
SH
Pantetheine
Cys
O
O
O
-O
Me
Me
P0 AT0
SH
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
S
S
ACP2 AT2
SH
Pantetheine
Cys
• KS1 catalyzes Claisen condensation
O
O
-O
CO2
O
Me
Me
P0 AT0
SH
POLYKETIDE BIOSYNTHESIS
ACP1 AT1 KS1 KR1 DH1 ER1
SH
S
ACP2 AT2
SH
Pantetheine
Cys
O
O
Me
P0 AT0
SHMe
Stereocenter
POLYKETIDE BIOSYNTHESIS
*
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 AT2
SH
Pantetheine
Cys
• KR1 catalyzes reduction of ketone
MeO O
SMe
P0 AT0
SH
POLYKETIDE BIOSYNTHESIS
*
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 AT2
SH
Pantetheine
Cys
SMe
O OH
Me
P0 AT0
SH
Stereocenter
POLYKETIDE BIOSYNTHESIS
**
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 AT2
SH
Pantetheine
Cys
• no DH1 activity
SMe
O OH
Me
P0 AT0
SH
POLYKETIDE BIOSYNTHESIS
**
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 AT2
SH
Pantetheine
Cys
• no ER1 activity
SMe
O OH
Me
P0 AT0
SH
POLYKETIDE BIOSYNTHESIS
**
ACP1 AT1 KS1 KR1 DH1 ER1
SH
ACP2 AT2 KS2
SH
SH
Pantetheine
Cys
• KS2 catalyzes translocation to module 2
KRCys
Me
O
HOMe
S
POLYKETIDE BIOSYNTHESIS
**
H1 ER1 ACP2 AT2 KS2
S
Pantetheine
KR2 DH2 ER2 TE
SH
CysOH
Ser
Me
O
HOMe
POLYKETIDE BIOSYNTHESIS
**
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)
O
CoAS
O
CoAS
O
O
3x CO2
3x
LNKS
mevinolin (=lovastatin®)
Me O
O
O O
OH
Me
MeO
SEnz(MeT)SAM
O
CoAS
O
O
3x CO2
3x
MeO
SEnz
Diels-Alder
MeOH
OH O
SEnz
O
CoAS
O
O
2x CO2
2x
MeOH
OH O
OH
P450
MeOH
OH O
OHOH
P450O
CoAS
O
CoAS
O
O
1x CO2
1x
LDKS(MeT)SAM
SEnz
OMe
nonaketide
diketide
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:
S
KSβ ACP
O
CoAS
O
O
O
CONH2
SSH
KSα ACP
O SHS
KSα ACP
O SSO O
O
O
SSH O
O
SSH OSHSO
O
CoAS
O
O
reduction(rarely)
KSα KSαACP ACP
ACPACP KSαKSα
translocationTE
n
ncycles
SHSO
O
OH KSα
n
ACP
decarboxylativeClaisen
condensation
KSβ = 'keto synthase β' (=decarboxylase!); KSα = 'keto synthase α' (=ketosynthase!); KR = keto reductase;DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein
1) ± KR2) ± DH3) ± ER
OOO
O
OO
Type II (iterative):
SH
KSβ ACP
CONH2
Biosynthesis of Actinorhodin – Type II PKS• actinorhodin – octaketide bacterial antibiotic (Streptomyces coelicolor)
– Hopwood Chem. Rev. 1997, 97, 2465 (DOI)
– timing of 1st cyclisation and mechanism of control of chain length uncertain• 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...
O
CoAS
O
O
8x CO2
7x
O
CoAS
O
O
O O O
O O
SEnz
O O
O 1
16
O O O
O
SEnz
O O
1
16
OHHO
KR
aromatase
cyclase
actinorhodin
O
OH
OH
O
O
CO2H2 H
O O O
O
SEnz
O O
O 1
16
OH
O O
O
SEnz
O O
1
16
OHO O
SEnz
O O
1
16
OH
OH
cyclase
octaketide
PKS[ACP, KSβ, KSα]
Scope of Structures - Type II PKS• microbial polyphenolic metabolites:
• many display interesting biological activities...
O
NH2
OOHOOH
OHHH
OHNMe2
Me
tetracycline
decaketides (10x C2)
pentaketides (5x C2) OMe
MeO OMe
O O
eugenone
hexaketides (6x C2)O
OOH
plumbagin
heptaketides (7x C2)
O
O
OHOH
HO
emodinoctaketides (8x C2)
nonaketides (9x C2)
O
OOHOH
MeO
rubrofusarin
OH
O
O OH
OOH
rabelomycine
Primary Metabolism - Overview
CO2 + H2O
1) 'light reactions': hv -> ATP and NADPH2) 'dark reactions': CO2 -> sugars (Calvin cycle)
OHOHOHOOH
HO
glucose& other 4,5,6 & 7 carbon sugars
Primary metabolism Secondary metabolites
oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)
phosphoenol pyruvate
glycolysis
CO2
HOOH
OHOOH
HO
POCO2
PO
erythrose-4-phosphate
SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans, f lavinoids+
shikimate
aromatic amino acids
aliphatic amino acidspeptidesproteinsCO2
Opyruvate
SCoA
Oacetyl coenzyme A
Citric acidcycle
(Krebs cycle)
ALKALOIDSpenicillinscephalosporinscyclic peptides
tetrapyrroles (porphyrins)
PHOTOSYNTHESIS
saturated fatty acidsunsaturated fatty acidslipids
FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
SCoA
OCO2
malonyl coenzyme A
CoAS
O
O
HO
HOCO2
mevalonateacetoacetyl coenzyme A
Primary metabolites
