Biosintesis Inglés Aagrasos

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?
– 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 OH
OH
OH
• Primary metabolites:
glycerol
– fully saturated, linear carboxylic acids & derived (poly)unsaturated derivatives:
3x fatty acids
• constituents of essential natural waxes, seed oils, glycerides (fats) & phospholipids
• structural role – glycerides & phospholipids are essential constituents of cell membranes OCOR1
OCOR2
• energy storage – glycerides (fats) can also be catabolised into acetate → citric acid cycle
OCOR3
• biosynthetic precursors – for elaboration to secondary metabolites glycerides
SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)]

e.g.
CO2H caprylic acid (C8, n = 2) CO2H myristic acid (C14, n = 5)
8 1 14 1
CO2H capric acid (C8, n = 3) CO2H palmitic acid (C16, n = 6)

1 16 1
10
CO2H lauric acid (C12, n = 4) CO2H stearic acid (C18, n = 7)

12 1 18 1
MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g. 9

9
CO2H CO2H
1
1
oleic acid (C18, Z-Δ )

9
palmitoleic acid (C16, Z -Δ )
9
18
16
(>80% of fat in olive oil )
POLY-UNSATURATED ACID DERIVATIVES (PUFAs)

e.g.
8 8 5
5 CO2H eicosapentaenoic acid (EPA)
CO2H arachidonic acid (AA)
1
1
(C20, Z -Δ5, Z -Δ8, Z -Δ11, Z -Δ14) (C20, Z-Δ5, Z -Δ8, Z-Δ11, Z -Δ14, Z -Δ17)
20
11 14 17 20 (in cod liver oil)
11 14
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 metabolism Primary metabolites Secondary metabolites
CO2 + H2O
PHOT OSY NT HESIS 1) 'light reactions': hv -> ATP and NADPH

2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO oligosaccharides
HOHO O
polysaccharides
HO nucleic acids ( RNA, DNA)
OH
glycolysis glucose
& other 4,5,6 & 7 carbon sugars
CO2
SHIKIMAT E METABOLIT ES
PO cinnamic acid derivatives
CO2 aromatic compounds
+ lignans
HO O HO OH
PO
OH OH
phosphoenol pyruvate erythrose-4-phosphate shikimate
ALKALOIDS
aromatic amino acids penicillins
peptides
CO2 cephalosporins
aliphatic amino acids proteins
cyclic peptides
O
pyruvate tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle) saturated f atty acids FAT T Y ACIDS & POLY KET IDES
SCoA SCoA unsaturated f atty acids polyacetylenes
CO2 lipids prostaglandins
O O aromatic compounds, polyphenols
acetyl coenzyme A malonyl coenzyme A macrolides
CoAS O HO ISOPRENOIDS
CO2 terpenoids
O HO steroids
mevalonate carotenoids
acetoacetyl coenzyme A
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
CO2
pyruvate GLYCOLYSIS
O
CoASH CO2
oxidative decarboxylation
NAD NADH
SCoA
acetyl CoA CITRIC ACID CYCLE
O
SCoA
acetyl CoA carboxylase
O CO2 (biotin-dependent)
CoASH
CoAS O
O saturated fatty acids FATTY ACIDS & POLYKETIDES

SCoA
acetoacetyl CoA unsaturated fatty acids prostaglandins
CO2 lipids
O polyacetylenes
2x NADPH SCoA 2x CoASH aromatic compounds, polyphenols
malonyl CoA
macrolides
O 2x NADP
HO ISOPRENOIDS
CO2 terpenoids
HO steroids
mevalonate (MVA) 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 Mg
Nu O O
Pi
O N NH
ATP H H O
NHEnz CoAS
S
O
enolate of acetyl CoA = Nu
O
Nu
O O HN NH
O O
ADP P H H
HO O OH NHEnz
O CoAS O
biotin S
carboxylase transcarboxylase
O
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 O
SEnz O O 2x NADPH
O O
EnzS O O O
O
CoAS EnzS
EnzS EnzS EnzS
CO2 2x NADP
EnzS
+ H2O
decarboxylative
dCc#1 reduction [R]#1
Claisen condensation dCc#2
[R]#3 dCc#3 [R]#2

fatty O O O O O O
acids
EnzS EnzS EnzS EnzS
The Decarboxylative Claisen Condensation (dCc)
• in vitro – the classical Claisen condensation:
EtO O O O O OEt O O
H
OEt OEt EtO EtO
+
O
EtO EtO
• in vivo - the decarboxylative Claisen condensation catalysed by a ketosynthase (KS)
O O
O O
SEnz O O
O EnzS
EnzS
O +
EnzS
EnzS
EnzS
CO2
– 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 O
O
H R cf . H R H R
H R S O O
S less ef f ective
3p 2p
pKa = 20 pKa = 25
– good leaving group ability of RS- (cf. RO-)

• due to pKa (RSH) ~10 cf. pKa (ROH) ~16
Nu: Nu:
O O Nu O O O Nu O
cf.
SR SR Nu OR
OR Nu
SR OR
a good leaving group 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)
ketone -> alcohol syn- alkene

O O reduction O OH O O
elimination hydrogenation O
EnzS EnzS EnzS
EnzS EnzS
HS
H
O O
HS H O H2O H HR O
NH2 NH2 NH2 NH2
N N N N
NADPH NADP NADPH 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
O S S O decarboxylative SH S O
O S SH CoAS O Claisen
condensation
O AT MT
KS ACP KS ACP KS ACP
CoAS
1) KR
reduction 2) DH
SH SH
3) ER
n = 2-8
cycles
KS ACP n
O S SH O S SH SH S O
O translocation
TE
n OH 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
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 N
O O
N N
HS N N O P O P O
H H HO O O O O O coenzyme A (CoASH)
H
pantetheinate chain HO P O OH
O O
ACP OH phpsphopantetheinyl transferase

(ACP synthase)
O O Me Me
HS O O ACP
N N P primed acyl carrier protein
H H -
O O
OH
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 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
FATTY ACID BIOSYNTHESIS (type II FAS)
ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

Cys
SH
Pantetheine
SH SH
NB. the following sequence of slides have been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
FATTY ACID BIOSYNTHESIS

Cys
SH
Pantetheine
O
Me S Co
SH SH
Acetyl-CoA
• AT1 loads acetyl group onto KS1


Cys
S
Pantetheine O
Me
SH SH

Cys
S
Pantetheine O
Me
SH SH
O O
-O S Co
Malonyl-CoA
• AT1 loads malonyl group onto ACP1


Cys
S
Pantetheine O
Me
S SH
O
O
O
-

Cys
S
Pantetheine O
Me
S CO2 SH
O
O
O
-
• KS1 catalyzes Claisen condensation


Cys
SH
Pantetheine
S SH
O
O
Me

Cys
SH
Pantetheine S Me
O O
SH
• KR1 catalyzes reduction of ketone


Cys
SH
Pantetheine S Me
O OH
SH

Cys
SH S Me
Pantetheine O OH
SH
• DH1 catalyzes dehydration of alcohol


Cys
SH S Me
Pantetheine O
SH

Cys
SH S Me
Pantetheine O
SH
• ER1 catalyzes reduction of alkene


Cys
SH S Me
Pantetheine O
SH
ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 KS2 KR

Cys Cys
SH SH
S
O
Pantetheine
Me
SH
• KS2 catalyzes translocation to module 2

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

Cys Ser
S OH
O
Pantetheine
Me
SH

Cys Ser
S OH
O
Pantetheine
Me
SH
O O
-O S Co
Malonyl-CoA
• MT2 loads malonyl group onto ACP2


Cys Ser
S OH
O
Pantetheine
Me
S
O
O
O
-

Cys Ser
S OH
O
Pantetheine
Me
S CO2
O
O
O
-


Cys Ser
SH OH
Pantetheine
S
O
O
Me

Cys Ser
SH S OH
Me
Pantetheine O O


Cys Ser
SH S OH
Me
Pantetheine O OH

Cys Ser
S Me
SH OH
Pantetheine
O OH
• DH2 catalyzes dehydration of alcohol


Cys Ser
S Me
SH OH
Pantetheine
O

Cys Ser
S Me
SH OH
Pantetheine
O
• ER2 catalyzes reduction of alkene


Cys Ser
S Me
SH OH
Pantetheine
O

Cys Ser
SH OH
S
Pantetheine O
Me
• TE catalyzes transesterification

Cys Ser
SH O
Pantetheine
O
SH
Me

Cys Ser
SH O H
O OH
Pantetheine
SH
Me
• TE catalyzes hydrolysis

Cys 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
O OH
5 O
CO2H CO2H CO2H
5-lipoxygenase H
H H
H
arachidonic acid leukotriene A4 (LTA4)
LTC4 synthase LTA4 hydrolase
OH OH OH
fatty acid CO2H CO2H
C5H11 S
O
H
NRPS H2N N CO2H leukotriene B4 (LTB4)
N
(glutathione) H
CO2H O
leukotriene C4 (LTC4)
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 O O
× (n+1)
CoAS O n SEnz n OH
FATTY ACIDS (FAs)

O
SCoA
O O O HO
O O
n SEnz CO2H
CoAS O × (n+1)
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 Me OMe O
HO OH O
O
O 2 O
O H
CO2H CO2H CO2H MeO O CO2H
OH OH OH Cl
OH O
6-methylsalicylic acid orsellinic acid citrinin Griseofulvin actinorhodin
(antibiotic) (kidney toxin (treatment for ring (antibiotic)
'yellow rice disease') worm infections)
O O
OMe
HO O O
H
OH O O
MeO POLYKETIDES
MeO O H
O O
aflatoxin B1
(mycotoxic carcingen)
N O O
O MeO
OH H OH
O HO O
O OH NMe2 Me
OH OH
HO O Me O
O OH O O O O
rapamycin O OH O O OMe
(immunosuppressant)
NB. a mixed polypropionate/acetate OH
O mevinolin
6-deoxyerythronolide B erythromycin A
NB. a polypropionate (antibiotic) (=lovastatin®)
(anti-cholesterol)
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 O O HO
OH O O OH
H2O OH
O OH O O O O O
2,4,6-triketone
dehydroacetic acid orcinol
(triketide)
– 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 HO OH
O HO
4x O O
OH OH OH OH
OH
O O O O 2x H2O OH O
14
3x H2O O O
C labelled
tetraketide
acetate
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 R = Me O O O O
• starter units: R = Et
CoAS R n i CoAS CoAS CoAS CoAS
R = Pr & Pr
acetyl CoA propionyl CoA butyryl CoA isobutyryl CoA
O O O O O O
O O
• extender units: R' = H
R' = Me CoAS O CoAS O CoAS O
CoAS O
R' = Et Me Et
R'
malonyl CoA (C2) Me-malonyl CoA (C3) Et-malonyl CoA (C4)
• non-functional or missing KR, DH, ER:

OH OH none H H
O (S) (Z) (E)
no KR no ER (R) no DH missing
or or
• 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 Nu = OH O
Nu = H2O O O O
intramolecular Nu = CoA NADH
EnzS O
HO CoAS 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)
Type I (modular & iterative): O O

O O
[Type II see later]
O S S O decarboxylative SH S O
O S SH CoAS O Claisen
condensation
O AT MT
KS ACP KS ACP KS ACP
CoAS
1) ± KR
reduction ? 2) ± DH
SH SH
3) ± ER
n
O cycles
KS ACP
O O O
n
O O O translocation
TE
n OH CE ACP KS 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
POLYKETIDE BIOSYNTHESIS [Type I – (modular)]
ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A

Cys
SH
Pantetheine Pantetheine Pantetheine
SH SH
NB. the following sequence of slides has also been adapted from: http://www.courses.fas.harvard.edu/%7echem27/
POLYKETIDE BIOSYNTHESIS

Cys
SH
SH SH
O
Me
S Co
Propionyl-CoA
• AT0 loads starting group (propionyl) onto ACP0


Cys
SH
S SH
O
Me

Cys
SH
Pantetheine Pantetheine S Pantetheine
O
Me
SH


Cys
S
Pantetheine Pantetheine O Pantetheine
Me
SH
SH
P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2

Cys
S
Pantetheine O
Me
SH SH
O O
SH
-O S Co
Me
Methylmalonyl-CoA
• AT1 loads methylmalonyl group onto ACP1


Cys
S
Pantetheine O
Me
S SH
O
Me
SH O
O
-

Cys
S
Pantetheine O
Me
CO2
S SH
O
Me
SH O
O
-


Cys
SH
Pantetheine
S SH
O * Me
SH O
Me Stereocenter

Cys
SH Me
Pantetheine S *
Me
O O
SH
SH


Cys
SH Me
Pantetheine S *
* Me
O OH
Stereocenter
SH
SH

Cys Me
SH
S *
Pantetheine * Me
O OH
SH
SH
• no DH1 activity

Cys Me
SH
S *
Pantetheine * Me
O OH
SH
SH
• no ER1 activity
ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 KS2 KR

Cys Cys
SH SH
S
O
Pantetheine * Me
HO *
Me
SH

H1 ER1 ACP2 AT2 KS2 KR2 DH2 ER2 TE

Cys Ser
S OH
Pantetheine
O
* Me
HO *
Me
SH
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 3x O O 2x O O
OH O
CoAS O CoAS O O O CoAS O
SEnz
Me Me
(MeT) SEnz SEnz Me
O LNKS SAM OH
Diels-
CoAS
Alder nonaketide
3x CO2 3x CO2 2x CO2
P450
O O OH O OH O
1x
CoAS O OH OH
Me OH Me
(MeT) OH P OH
O 450
LDKS SAM O OH
Me
CoAS O
SEnz Me
1x CO2
Me O
O O
diketide
mevinolin (=lovastatin®)
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 O O
Type II (iterative): O O
S O SH S O O S SH O S S O decarboxylative SH S O
CoAS O Claisen
O O condensation
KSβ ACP KSα ACP KSα ACP KSα ACP KSα ACP
CoAS O
1) ± KR
CONH2 reduction 2) ± DH
(rarely)
3) ± ER
SH
n
O
cycles
KSβ ACP O n O O
O O O translocation
TE
n OH 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
Biosynthesis of Actinorhodin – Type II PKS
• actinorhodin – octaketide bacterial antibiotic (Streptomyces coelicolor)
– Hopwood Chem. Rev. 1997, 97, 2465 (DOI)
7x O O
CoAS O
O O O O O O O O O
PKS
O O 16
[ACP, KSβ , KSα]
16 16
O O O O cyclase O O O KR O O O
CoAS O
O 1 SEnz O 1 SEnz HO 1 SEnz
OH OH
8x CO2 octaketide
aromatase
OH O OH O O OH O O
16 16
O cyclase
2 O O
H O O O
CO2H
1 SEnz 1 SEnz
OH O OH
actinorhodin
– 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...
Scope of Structures - Type II PKS
• microbial polyphenolic metabolites:
OH O OH
pentaketides (5x C2) OMe O O octaketides (8x C2)
emodin
eugenone
HO
MeO OMe
O
hexaketides (6x C2) nonaketides (9x C2) OH O OH O O

O
NH2
plumbagin tetracycline
OH
Me H H
OH NMe2
OH O
O
O
heptaketides (7x C2) MeO O OH
decaketides (10x C2)
rubrofusarin rabelomycine
OH OH O
OH O OH
• many display interesting biological activities...

Primary Metabolism - Overview
Primary metabolism Primary metabolites Secondary metabolites
CO2 + H2O
PHOT OSY NTHESIS 1) 'light reactions': hv -> AT P and NADPH

2) 'dark reactions': CO2 -> sugars (Calvin cycle)
HO O oligosaccharides
HOHO polysaccharides
HO nucleic acids ( RNA, DNA)
OH
glycolysis glucose
& other 4,5,6 & 7 carbon sugars
CO2
SHIKIMAT E METABOLIT ES
PO cinnamic acid derivatives
CO2 aromatic compounds
+ lignans, f lavinoids
HO O HO OH
PO
OH OH
phosphoenol pyruvate erythrose-4-phosphate shikimate
ALKALOIDS
aromatic amino acids penicillins
peptides
CO2 cephalosporins
aliphatic amino acids proteins
cyclic peptides
O
pyruvate tetrapyrroles (porphyrins)
Citric acid
cycle
(Krebs cycle) saturated f atty acids FAT T Y ACIDS & POLY KET IDES
SCoA SCoA unsaturated f atty acids prostaglandins
CO2 lipids polyacetylenes
O O aromatic compounds, polyphenols
acetyl coenzyme A malonyl coenzyme A macrolides
CoAS O HO ISOPRENOIDS
CO2 terpenoids
O HO steroids
mevalonate carotenoids
acetoacetyl coenzyme A

Biosintesis Inglés Aagrasos

Enviado por

Dados do documento

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

Biosintesis Inglés Aagrasos

Enviado por

Direitos autorais:

Formatos disponíveis

Biosynthesis – Inspiration for Drug Discovery

Biosynthesis of Fatty Acids & Polyketides

SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)]

CO2H capric acid (C8, n = 3) CO2H palmitic acid (C16, n = 6)

CO2H lauric acid (C12, n = 4) CO2H stearic acid (C18, n = 7)

MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g. 9

oleic acid (C18, Z-Δ )

POLY-UNSATURATED ACID DERIVATIVES (PUFAs)

PHOT OSY NT HESIS 1) 'light reactions': hv -> ATP and NADPH

O saturated fatty acids FATTY ACIDS & POLYKETIDES

[R]#3 dCc#3 [R]#2

• in vivo - the decarboxylative Claisen condensation catalysed by a ketosynthase (KS)

weak C-S bond

– good leaving group ability of RS- (cf. RO-)

1. NADPH-mediated ketone → alcohol reduction catalysed by a keto reductase (KR)

ketone -> alcohol syn- alkene

NADPH NADP NADPH NADP

ACP OH phpsphopantetheinyl transferase

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• AT1 loads acetyl group onto KS1

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• AT1 loads malonyl group onto ACP1

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• KS1 catalyzes Claisen condensation

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• KR1 catalyzes reduction of ketone

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• DH1 catalyzes dehydration of alcohol

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

• ER1 catalyzes reduction of alkene

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 KS2 KR

• KS2 catalyzes translocation to module 2

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

• MT2 loads malonyl group onto ACP2

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

• KS2 catalyzes Claisen condensation

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

• KR2 catalyzes reduction of ketone

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

• DH2 catalyzes dehydration of alcohol

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

• ER2 catalyzes reduction of alkene

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE

arachidonic acid leukotriene A4 (LTA4)

LTC4 synthase LTA4 hydrolase

FATTY ACIDS (FAs)

the polyketide pathway

– this leads to enormous diversity...