3.0 Fatty Acids and Polyketides : Biosynthesis and Engineering

Transcription

1 3.0 Fatty Acids and Polyketides : Biosynthesis and Engineering Chemical tructure Chemical Formula: C Palmitic Acid = LAD aponification Na Na = AP Present in animal and plant fats as triacyl glycerols Isotopic Feeding Experiments eveal: The Biosynthesis of Unsaturated Fatty Acids 2 Desaturase enzyme Palmitic acid Palmitoleic acid 'cis' olefins are characteristic of desaturases

2 3.1 The Biosynthesis of Fatty Acids 3.1A The Proteins 3.1B The Chemical eactions Crystal tructure of the mamallian Fatty Acid ynthase (FA) CoA CoA K K + nb - The and don't show up in the crystal structure "Cartoon" View EPE CYCLE The tarter Unit C 2 K NADP The Extender Unit 2 NADP K = Ketoynthase = Ketoeductase = Deydratase = Enoyl eductase = Acyl Transferase = Acyl Carrier Protein = Thiolesterase 2 When chain is 16 or 18 carbons

3 CoA loads starter unit epeat Cycle transfers starter to K loads Extender CoA transfers extender to,, Acyl K ready for another round of extension Acyl group transfers back to K Acyl group resides on for subsequent processing K and catalyse chain extension Acyl group transfers to when it is the correct length Nu Nu releases acyl group using a suitable nucleophile (could be water!)

4 3.2 Comparing Polyketides and Fatty Acids Polyketide chains are formed by the head-to-tail assembly of carboxylic acids AM N 2 acetate propionate hexanoate malonamide Notes TAIL EAD erythromycin heptaketide EAD TAIL TAIL EAD diketide EAD TAIL lovastatin nonaketide TAIL N 2 Polyketide biosynthesis always starts with a 'starter group' (red) - this forms the tail of the final polyketide. The extender units are arranged in a head (C1) to tail (C2) direction. Pendant methyl groups can come from propionate (usually bacterial polyketides) or from the methyl of AM (usually fungal polyketides). The length of a polyketide is indicated by the number of building blocks used - e.g. diketide, triketide etc. Lovastatin is formed from two chains, a diketide and a nonaketide. norsolorinic acid octaketide oxytetracycline nonaketide EAD

5 3.3 A closer Look at Polyketides Examples Me Erythromycin A accharopolyspora erythrae (bacterium) Antibiotic N Isotopic Feeding Experiments show skeletons derived from simple carboxylic acids AM N 2 acetate propionate hexanoate malonamide N Me NMe 2 N 2 Lovastatin Aspergillus terreus (fungus) Cholesterol biosynthesis inhibitor (MG-CoA reductase) Norsolorinic acid Me Aflatoxin B 1 Aspergillus parasiticus (fungus) potent liver carcinogen NMe 2 N 2 xytetracycline treptomyces rimosus (bacterium) antibiotic

6 3.4 Architectures of Polyketide ynthases Type I systems - multifunctional Proteins 3.4.1A Classic Modular PK - e.g. deoxyerythronolide synthase (DEB) Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 6-deoxy erythronolide-b (6-DEB) Everything required contained in modules - each module must have a minimum of K, and s can select different extender units in each module - although here they are all selective for methylmalonyl CoA 3.4.1B Trans- Modular PK 1.!Cortes, J., aydock,. F., oberts, G. A., Bevitt, D. J., and Leadlay, P. F. (1990) An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of accharopolyspora erythraea., Nature 348, CMeT CMeT K 0 K K K K 0 K K C EC1 EC2 Modules generally lack trans-acting components - malonate only extender But β-methyls can come via CMeT domain other trans-acting components including C cassette ome domains are non-functional ( 0 ) ome modules are mysterious - K 0 - load / transfer? X Mupirocin precursor X C EC1 EC2 X X C Chemistry 1.!El-ayed, A. K., othersall, J., Cooper,. M., tephens, E., impson, T. J., and Thomas, C. M. (2003) Characterization of the Mupirocin Biosynthesis Gene Cluster from Pseudomonas fluorescens NCIMB 10586, Chemistry & Biology 10,

7 3.4 Architectures of Polyketide ynthases 3.4.1C Iterative Fungal PK (ipk) - single modules act several times in a prgrammed way - different chemistry in each cycle 3.4.1C-1 Non-educing ipk (N-iPK) 3.4.1C-2 Partially educing ipk (P-iPK) PT CMeT X [] CE (tarter Unit Acyl Transferase) domain loads starter unit (acetate here) K,, extend PT (product template) cyclises Various processing and release options - here CMeT and reductive release nly known to make 6 methyl salicylic acid 3.4.1C-3 ighly educing ipk (-ipk) - like a full module of a modular system, but iterative. Different chemistry in each cycle CMeT CMeT 0 Variations - e.g. -ipk-np K CMeT 0 C A PCP DKC ome have functional e.g. squalestatin tetraketide synthase (QTK) ome have broken and a trans-acting, e.g. lovastatin nonaketide synthase (LNK) N The tenellin synthetase links a polyketide to phenylalanine and uses a Dieckmann ynthase (DKC) to form a 5-membered heterocycle.

8 3.4 Architectures of Polyketide ynthases Type II ystems, e.g. actinorhodin (act) synthase K α K β "Minimal PK" + CoA Loading CoA x 7 K β Initiation C 2 x 1 Discrete monofunctional proteins No needed - loads itself Add more proteins - get more functions K α Extension -bound Intermediate 2 Actinorhodin (model system) Many teps Aro Cyc Cyclisation and elease EK4 EK4b Type III ystems, e.g. Chalcone ynthase + CoA CoA K Enz Enzyme-Bound Intermediate Chalcone Naringenin

9 3.5 The apamycin ynthase Load CL p p p a 0 a 0 p p a a p a p a a N rapqnml Tailoring Me N Me Me apamycin 0 p Domains must be inactive - other processing domains may or may not be functional Domains selective for methylmalonyl CoA a Domains selective for malonyl CoA CL Loads a special starter unit 1.!Aparicio, J., Molnar, I., chwecke, T., Konig, A., aydock,., Khaw, L., taunton, J., and Leadlay, P. F. (1996) rganization of the biosynthetic gene cluster for rapamycin in treptomyces hygroscopicus: Analysis of the enzymatic domains in the modular polyketide synthase, Gene 169, 9 16.

10 3.6 Engineering DEB 1.!Xue, Q., Ashley, G., utchinson, C.., and anti, D. V. (1999) A multiplasmid approach to preparing large libraries o polyketides., P Natl Acad ci Usa 96, Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 Wild Type Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 emoval of a function -- replaced by -LINK- Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 Addition of a function Module 2 to - Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 Engineering in Module 2 and module 5

11 3.6 Engineering DEB Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 6-deoxy erythronolide-b (6-DEB) Move from module 6 to module 2 DEB1-1.!Cortés, J., Wiesmann, K., oberts, G., Brown, M., taunton, J., and Leadlay, P. (1995) epositioning of a domain in a modular polyketide synthase to promote specific chain cleavage, cience 268, a Introduce from apamycin module 2 1.!liynyk, M., Brown, M. J., Cortes, J., taunton, J., and Leadlay, P. F. (1996) A hybrid modular polyketide synthase obtained by domain swapping., Chemistry & Biology 3, a Tailoring n.b. eryk and eryg don't work! N 1.!Petkovic,., Lill,. E., heridan,. M., Wilkinson, B., McCMICK, E. L., McATU,. A., taunton, J., Leadlay, P. F., and Kendrew,. G. (2003) A novel erythromycin, 6-desmethyl erythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase, J Antibiot, Nature Publishing Group 56,

12 3.7 Avermectin Biosynthesis Me Me Loading Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 Avermectin A1a Module 7 Module 8 Module 9 Module 10 Module 11 Module 12 Me 1 Me Glycosyl Transfer Me ing Formation 19 Probable P Me ing Formation eduction + -methylation Post PK Tailoring 1 First enzyme free Intermediate 19

13 3.8 ENGINEING to make DAMECTIN N 2 Loading Module 1 Module 2 Module 3 Module 4 Module 5 Module 6 P Phoslactamycin B Loading Module 1 Module 2 Cyclohexane carboxylic acid elective Loading Module Module 7 Module 8 Module 9 Module 10 Module 11 Module 12 1 Me Me 19 Doramectin used in Veterinary Medicine 1 19 Post PK Tailoring 1 First enzyme free Intermediate Me 19

14 3.9 Iterative Type I PK KEY Proteins - ITIVE not Modular CMeT LovB CMeT LovF LovC Post-PK steps Lovastatin Aspergillus terreus (fungus) Cholesterol biosynthesis inhibitor (MG-CoA reductase) Diels Alder elease to Lactone 8 extensions 1 protein different rxns each time! Methylation Mechanism 2 x [] Further Extensions Direct Acyl Transfer from LovB Me Ad Me AM is a biological "MeI" N 2