Biosynthesis of Natural Products

Home , Fatty acid synthesis, Protein complex

Biosynthesis of Fatty Acids & Polyketides

Nov 2019 Format & Scope of Lecture

• 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 • Primary metabolites: OH 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 • energy storage – glycerides (fats) can also be catabolised into acetate → citric acid cycle OCOR2 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

CO H CO2H capric acid (C8, n = 3) 2 palmitic acid (C16, n = 6) 1 10 1 16

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 CO H CO2H 2 1 1 9 oleic acid (C18, Z-9) palmitoleic acid (C16, Z - ) 18 16 (>80% of fat in olive oil )

POLY-UNSATURATED ACID DERIVATIVES (PUFAs) e.g. 8 5 8 5 CO2H eicosapentaenoic acid (EPA) CO2H arachidonic acid (AA) 1 5 8 11 14, 17 1 (C20, Z -5, Z -8, Z -11, Z -14) (C20, Z- , Z - , Z- , Z - Z - ) 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 1) 'light reactions': hv -> ATP and NADPH PHOTOSYNTHESIS 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 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated f atty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated f atty acids prostaglandins lipids CO2 polyacetylenes O O aromatic compounds, polyphenols acetyl coenzyme A malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate

For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html 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 CO O CoASH 2 (biotin-dependent) CoAS O

saturated fatty acids O SCoA FATTY ACIDS & POLYKETIDES acetoacetyl CoA unsaturated fatty acids prostaglandins CO O 2 lipids polyacetylenes 2x CoASH 2x NADPH SCoA malonyl CoA aromatic compounds, polyphenols 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 O NHEnz CoAS O biotin S transcarboxylase carboxylase 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): 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 OO 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  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 *C=O; (i.e. minimal ‘resonance’ nS → *C=O)

polarised reacive carbonyl

weak C-S bond

O O O O H R cf . H R H R H R S O S less ef f ective O 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- OO alkene reduction OOH elimination O hydrogenation O O EnzS EnzS EnzS EnzS EnzS HS H O O H O O O HS H 2 H HR NH2 NH NH2 NH2 2

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 decarboxylative SHS O O S SH OOS S CoAS O Claisen condensation O AT MT KS ACP KS ACP CoAS KS ACP

1) KR reduction 2) DH SH SH 3) ER n = 2-8 cycles KS ACP n

O S SH O S SH SHS 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 O HO H O O O O coenzyme A (CoASH)

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 - OH 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 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

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine

O Me S Co SH SH Acetyl-CoA

•AT1 loads acetyl group onto KS1 FATTY ACID BIOSYNTHESIS

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

SH SH FATTY ACID BIOSYNTHESIS

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

SH SH O O - O S Co Malonyl-CoA

•AT1 loads malonyl group onto ACP1 FATTY ACID BIOSYNTHESIS

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

S SH O O O- FATTY ACID BIOSYNTHESIS

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

S CO2 SH O O O-

•KS1 catalyzes Claisen condensation FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine

S SH O O Me FATTY ACID BIOSYNTHESIS

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

•KR1 catalyzes reduction of ketone FATTY ACID BIOSYNTHESIS

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

SH FATTY ACID BIOSYNTHESIS

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

•DH1 catalyzes dehydration of alcohol FATTY ACID BIOSYNTHESIS

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

SH FATTY ACID BIOSYNTHESIS

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

•ER1 catalyzes reduction of alkene FATTY ACID BIOSYNTHESIS

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

SH FATTY ACID BIOSYNTHESIS

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 FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

SH FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

SH O O - O S Co Malonyl-CoA

•MT2 loads malonyl group onto ACP2 FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

S O O O- FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

S CO2 O O O-

•KS2 catalyzes Claisen condensation FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH OH

Pantetheine

S O O

Me FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH S Me OH Pantetheine O O

•KR2 catalyzes reduction of ketone FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH S Me OH Pantetheine O OH FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O OH

•DH2 catalyzes dehydration of alcohol FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O

•ER2 catalyzes reduction of alkene FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH OH S Pantetheine O

• TE catalyzes transesterification FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH O

Pantetheine O SH

Me FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH O H OH Pantetheine O SH

• TE catalyzes hydrolysis FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE 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 (

• leukotrienes are the other main class of 2° metabolites derived from arachiodonic acid – they are potent (

OO OOH 5 O CO2H CO2H CO2H 5-lipoxygenase H HH H

arachidonic acid leukotriene A4 (LTA4)

LTC4 synthase LTA4 hydrolase

OH OH OH

fatty acid CO2H CO2H

S C5H11 O H NRPS H2N NCO2H 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 synthesised in a very similar fashion. The difference is that during the iterative cycle of chain extension the -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

OOO HO O O SEnz CoAS O × (n+1) n 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 Me OMe O OH O HO O O O O 2 CO H CO H O H 2 2 CO2H MeO 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 O HO O H OH O O MeO POLYKETIDES O H O O MeO aflatoxin B1 (mycotoxic carcingen) O O N O MeO OH H OH O HO O O OH NMe OH OH 2 Me HO O Me O O OH O O OO

rapamycin OOH O O OMe (immunosuppressant) NB. a mixed polypropionate/acetate O OH 6-deoxyerythronolide B erythromycin A mevinolin 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 O OH O O O O O OH 2,4,6-triketone dehydroacetic acid (triketide) 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 OH O O HO 4x O O OH OH OH OH OH OO 2x H O OH O 3x H2O OO OO 2 14C 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 -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 CoAS CoAS CoAS CoAS R = nPr & iPr acetyl CoA propionyl CoA butyryl CoA isobutyryl CoA

O O O O O O O O • extender units: R' = H CoAS O CoAS O R' = Me CoAS O CoAS O R' = Et R' Me Et malonyl CoA (C2) Me-malonyl CoA (C3) Et-malonyl CoA (C4) • non-functional or missing KR, DH, ER:

OH OH none O (S) (Z) (E) HH 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:

Nu = OH O O Nu = H O O O 2 intramolecular Nu = CoA NADH O 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] decarboxylative SHS O O S SH OOS S CoAS O Claisen condensation O AT MT KS ACP KS ACP CoAS KS ACP

1) ± KR reduction ? 2) ± DH SH SH 3) ± ER n O cycles KS ACP O O n O O S SH O S SH SHS O 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

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine Pantetheine

SH SH O Me S Co Propionyl-CoA

•AT0 loads starting group (propionyl) onto ACP0 POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine Pantetheine

S SH O

Me POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine S Pantetheine O

•KS1 catalyzes translocation to module 1 POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys S Pantetheine Pantetheine O Pantetheine

SH SH POLYKETIDE BIOSYNTHESIS

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

SH SH O O SH - O S Co Me Methylmalonyl-CoA

•AT1 loads methylmalonyl group onto ACP1 POLYKETIDE BIOSYNTHESIS

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

S SH O Me SH O O- POLYKETIDE BIOSYNTHESIS

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

CO S 2 SH O Me SH O O-

•KS1 catalyzes Claisen condensation POLYKETIDE BIOSYNTHESIS

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

S SH O * Me SH O

Me Stereocenter POLYKETIDE BIOSYNTHESIS

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

•KR1 catalyzes reduction of ketone POLYKETIDE BIOSYNTHESIS

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

Stereocenter SH

SH POLYKETIDE BIOSYNTHESIS

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

•no DH1 activity POLYKETIDE BIOSYNTHESIS

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

•no ER1 activity POLYKETIDE BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 KS2 KR Cys Cys SH SH S O Pantetheine * Me HO * Me

•KS2 catalyzes translocation to module 2 POLYKETIDE BIOSYNTHESIS

H1 ER1 ACP2 AT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine * 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)

DEBS1 DEBS2 DEBS3

module 1 module 2 module 3 module 4 module 5 module 6 O

KR KR DH ER KR KR KR AT KS AT KS AT KS AT KS AT KS AT KS AT TE OH release S S S S S S S O OH O O O O O O O O OH HO HO O HO HO

HO HO O HO loading O HO HO O

HO HO O OH OH HO HO O OH

HO O OH = Acyl Carrier Protein erythronolide B TE = Thioesterase 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 O O O O O 3x 3x 2x 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 OH O OH O 1x O O CoAS O OH OH Me OH Me (MeT) OH OH P450 O LDKS SAM O OH Me CoAS O SEnz Me 1x CO 2 Me O OO 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, KS, KS, [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’  (KS) to give S-acetyl-ACP

– the extender malonates undergo decarboxylative Claisen condensations by ketosynthase  (KS) • these clusters rarely utilise KR, DH or ER activities and produce ‘true’ polyketides:

O O Type II (iterative): CONH2 O O S O SH S O O S SH OOS S decarboxylative SHS O CoAS O Claisen O O condensation KS CoAS O KS ACP KS ACP KS ACP ACP KS ACP

1) ± KR CONH2 reduction 2) ± DH (rarely) 3) ± ER SH n O cycles

KS ACP O n O O O S SH O S SH SHS O O O O translocation TE n OH KS ACP KS ACP KS ACP

KS = 'keto synthase ' (=decarboxylase!); KS = 'keto synthase ' (=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 OOO OOO OOO PKS 16 O O [ACP, KS, KS] 16 16 OOOOcyclase O OO KR O OO CoAS O O 1 SEnz O SEnz HO SEnz OH 1 OH 1 8x CO2 octaketide aromatase

OH O OH OO OH OO 16 16 O cyclase 2 H OO O OO CO2H 1 SEnz SEnz OH O OH 1 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 OO octaketides (8x C2) emodin eugenone HO MeOOMe 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 OOH

• many display interesting biological activities... Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 +H2O 1) 'light reactions': hv -> ATP and NADPH PHOTOSYNTHESIS 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 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 aromatic compounds + HO HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

CoAS O HO ISOPRENOIDS terpenoids CO2 O HO steroids carotenoids acetoacetyl coenzyme A mevalonate

For interesting animations’ of e.g. photosynthesis see: http://www.johnkyrk.com/index.html