Biosynthesis of Natural Products Biosynthesis of Fatty Acids & Polyketides Alan C. Spivey [email protected] Nov 2015 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 • 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): Malonyl CoA O O SEnz O O 2x NADPH O O EnzS OO O O CoAS EnzS EnzS EnzS EnzS CO 2x NADP 2 EnzS + H2O decarboxylative #1 #1 dCc reduction [R] #2 Claisen condensation dCc [R]#3 #3 [R]#2 fatty O OO dCc O OO 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 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 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).