Lecture 1 hemical classes of secondary metabolites & Identification and functional analysis of biosynthetic gene clusters ne third of top selling pharmaceuticals are produced from plants or microrganisms Lutz eide Pharmaceutical Institute Tübingen University Germany Taxus sp. 3 3 treptomyces sp. MedIL plit 27: Microbial Genomics and econdary Metabolism Paclitaxel Vancomycin tructure Most frequently prescribed classes of antibiotics (Germany, 25) 1. hemical classes of secondary metabolites 2. Identification and functional analysis of biosynthetic gene clusters hemical classes of antibiotics β-lactam antibiotics (Penicillins, Aminopenicillins, ephalosporins) Peptide antibiotics Glykopeptide antibiotics Lipopeptide antibiotics Fluorquinolones Aminomarins Tetracyclines Makrolides Aminoglycosides Lincosamides xazolidinones ulfonamides itroimidazoles etc. Red = synthetic substances Functional classes of antibiotics biosynthese Bacitracin Vancomycin Mechanism of action blue = antimycotics xazolidinones Daptomycin
Biosynthetic classes of antibiotics Polyketide antibiotics (1): Tetracyclines Polyketides (Tetracyclines, Macrolides) on-ribosomal peptides and amino acid derivatives (peptide antibiotics, ß-lactam antibiotics) ligosaccharides (Aminoglycosides) Isoprenoids (Fusidic acid), Meroterpenoids, other isoprenylated componds etc. 7 8 9 1 6 5 11 12 4 1 2 natural compounds from treptomyces semisynthetic Polyketide antibiotics (2): Macrolides Erythromycin and semisynthetic derivatives Amino sugar semisynthetic Lactone Desoxy sugar semisynthetic Roxithromycin Erythromycin A 14-membered lactone Tylosin 16-membered lactone Erythromycin A accharopolyspora erythrea (=treptomyces erythraeus) larithromycin Macrolides: Biosynthesis by modular polyketide synthases Polyketide antibiotics (3): Ansamacrolides Lactam Rifamycin B from Amycolatopsis mediterranei semisynthetic semisynthetic Rifamycin V Rifampicin
Rifamycin biosynthesis by modular polyketide synthase starter unit: 3-amino-5-hydroxybenzoic acid Rif rf 19 on-ribosomal peptides Rifamycine: : Biosynthese from Amycolatopsis orientalis (= treptomyces orientalis) on-ribosomal peptides Biosynthetic gene cluster of DA 2 R DA = alcium-dependent antibiotic from treptomyces coelicolor A3(2) R A T A T -AMP -AMP - - 2 2 2-2 2 R 2 R Amino acid Resistance Regulation synthesis 2kb 82kb 24kb cdap1 cdap2 cdap3 64kb non-ribosomal 2kb peptide synthetases Fatty acid synthesis cdap1 A T A T A T E A T A T A T E M1 (er) M2 (Thr) M3 (Trp) M4 (Asp) M5 (Asp) M6 (PG) DA cdap2 A T A T A T E cdap3 A T A T TE M7 (Asp) M8 (Gly) M9 (Asn) M1 (Glu/Melu) M11 (Trp) A: Adenylatiom domain; T: Thiolation domain ; : condensation domain; E: epimerization domain; TE: thioesterase domain Bacitracin Daptomycin (ubicin ): a lipopeptide fatty acid Bacitracin A from Bacillus licheniformis: a dodeka-peptide Leucine ysteine D-Glutamic L-LysineLysine acid α ε L-LeucineLeucine L-Isoleucine D-rnithine L-Asparagine D-Asparagic acid L-Isoleucine D-Phenylalanine L-istidine from treptomyces roseosporus
ß-Lactams: biosynthesis of penicillins und cephalosporins 2 2 + + 3 2 L-α-Aminoadipinsäure L-ystein L-Valin = A = = V 2 Isopenicillin 1. AV ynthetase: LLL-AV-Tripeptide 2. AV Racemase: LLD-AV-Tripeptid 3. AV yclase = Isopenicillin -ynthase Acyl-oA:IP Acyltransferase (Penicillin-Acylase) 2 3 6-Aminopenicillic acid Biosynthesis of Penicillin G 2 Acyl-oA:IP Acyltransferase (Penicillin-Acylase) Isopenicillin 2 3 3 oa 2 Benzylpenicillin = Penicillin G 3 3 Biosynthesis of cephalosporins ligosaccharides: Aminoglycosides 2 * Isopenicillin inversion at α- of AA ring expansion hydroxylation acetylation 2 2 ephalosporin similar: ephamycin from treptomyces sp. Acremonium strictum (ephalosporium acremonium), Ascomycetes Aminocyclitoles: 2 2 2 Desoxy- deoxy streptamine 2 2 2 2 streptamine treptamin 2 2 plus sugars and aminosugars 2 streptidine treptidin 2 2 Aminoglycosides Meroterpenoids: Polyketide/isoprenoid hybrid compounds treptomycin Kanamycin 3 3 eomycin 2 aphterpin from aus treptomyces sp. strain L19 (oel, alk Institute) Furanonaphthoquinone I from treptomyces sp. Gentamicin
on-isoprenoid/isoprenoid hybrids ther structural classes 3 hloramphenicol 2 (l) Lincomycin ovobiocin (an aminomarin antibiotic) Endophenazine A tructure econdary metabolic genes in plant and microbial genomes 1. hemical classes of secondary metabolites Plant genome: Bacterial genome: 2. Identification and functional analysis of biosynthetic gene clusters Biosynthetic genes: scattered over the genome Gene cluster Biosynthetic genes: combined in a cluster oumarin antibiotics: tructural similarities and differences oumarin antibiotics: Inhibitors of gyrase and topoisomerase IV 2 ovobiocin 3 l lorobiocin gyrase GyrA GyrA Fluoroquinolones topoisomerase IV Par Par 3 3 3 oumermycin A1 3 3 GyrB Function: GyrB oumarins k D 1 nm introduction of negative supercoils control of DA supercoiling ParE Function: ParE decatenation of daughter chromosomes
Biosynthesis: Previous knowledge Deoxysugar Interaction of obiocin with the B subunit of bacterial gyrase Aminomarin ddp-glucose- 4,6-dehydratase Glucose 2 -Adenosyl-methionine Tyrosine?? ovobiocin Tyrosine*?? * resp. 4--phenylpyruvate Pojer et al. JB 278:3661 (23) creening of cosmid bank creening der osmid-genbank 1 2 3 4 5 6 7 8 9 1 11 12 A B D E F G A B total 98 cosmids: twenty pools of 48 nes each A B D E F G 1 2 3 4 5 6 7 8 9 1 11 12 A B pools of 8 nes single nes creening with dehydratase probe loning of the gene clusters by screening with a ddp-glucose 4,6-dehydratase probe Expect: 4-5 positive cosmids (estimated from genome size = 8.5 Mb and 98 cosmids) found: 4 positive cosmids ovobiocin cluster lorobiocin cluster Probe T gyrb R T gyrb R oumermycin A1 cluster T gyrb R
loning of the biosynthetic gene clusters Deoxysugar biosynthesis 2 ovobiocin 3 3 l lorobiocin 2 dtdp-1-glucose synthase 2 dtdp-glucose 4,6-dehydratase dtdp-4-keto- 6-deoxyglucose 3,5 -epimerase - P 3 ovv dtdp ovt dtdp ovw Glucose-1-phosphate 3 3 3 3 3 oumermycin A1 3 3 -Methyltransferase dtdp ovu dtdp-4-keto- 6-desoxy-hexose reductase dtdp ov dtdp 22 2 2 EF G I JK L M PQRT UV WgyrBR E F G Y I JKLM 1 23 4 5 6 7 halp QR TUVWZ gyrb R pary R pary E G Y I JKLM P R R R R R R gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R R T UVWgyrB R T UVW gyrb R T UVWgyrB R absorbance (UV 35 nm) Inactivation of T : PL examination.4.3.2.1 Wild-type ovobiocin 1 2 T mutant 3 3 3 3 2 -Methylation of iose 3 l ovobiocin lorobiocin 2 3 3 3 3 3 3 3 3 3 3 3 oumermycin A1 3. 15 2 time (min) P P P T UVWgyrB R T UVW gyrb R T UVWgyrB R Inactivation of P : PL examination Biosynthesis of aminomarin ring Wild-type oumermycin A1 M- = 118 2 Tyrosine ov ATP 2 ovi 2 ov P 45 ov P mutant Bis-desmethyl- oumermycin A1 M- = 18 ovj ydroxylation ovk??? 2 ov 2 lo-al R R=-3 R = -l EF G IJKL M PQRT UV WgyrB R E F G Y IJKLM 1 23 4 5 6 7 halp QR TUVWZ gyrb R pary R E G Y IJKLM P R RRR RR gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R pary R
FRT FRT FRT FRT Inactivation of hal by PR targeting Inactivation of hal: PL analysis ~2,5 kb ~2,5 kb wild-type cosmid D1A8-h-773 integration mutant probe Eco19I Eco19I start hal Eco19I start hal Eco19I hal 14 bp orit orit Paac neo Paac Eco19I aac(3)iv 1662 bp stop hal double crossover Eco19I aac(3)iv stop hal 8576 bp 7427 bp 616 bp 4899 bp 3639 bp 2799 bp 1953 bp 1882 bp 1515 bp 1482 bp 1164 bp 992 bp MWT 1 2 3 outhern Blot analysis. WT = wild-type 1, 2, 3 = hal - mutant 3 3 hal - mutant [M-] - : m/z = 661.3 3 ovbiocin 11 35-45 µg/ml 3 Wild type [M-] - : m/z = 695. l lorobiocin 2-25 µg/ml Gust et al., PA 1:1541 (23) 1662 bp Eustaquio et al. hem Biol 1:279 (23) Eustaquio et al. hem Biol 1:279 (23) Expression of the methyl transferase ov in the hal - mutant Biosynthesis of prenylated benzoate moiety 3 3 hal - mutant [M-] - : m/z = 661.3 hal - mutant + empty vector ovbiocin 11 35-45 µg/ml 3 hal - mutant + vector with [M-] - : m/z = 675. 3 ovbiocin 12 28-58 µg/ml Eustaquio et al. hem Biol 1:279 (23) l ovobiocin lorobiocin 2 3 3 3 3 3 3 3 3 oumermycin A1 E F G I JK L M PQRT UV WgyrB R E F G Y I JKLM 1 23 4 5 6 7 halp QR T U VW Z gyrb R pary R E G Y I JKLM P R RRR RR gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R pary R Biosynthesis of prenylated benzoate moiety: ypothesis 1 Biosynthesis of prenylated benzoate moiety: ypothesis 1 2 Tyr 3 - lyase ß-oxidation prenyl transferase 2 Tyr 3 - lyase ß-oxidation prenyl transferase Tyrosine 4-oumarate Prenylated 4-hydroxybenzoate () Tyrosine 4-oumarate 4-ydroxybenzoate 4-ydroxybenzoate Prenylated 4-hydroxybenzoate () similar to: Brad Moore and coworkers Enterocin biosynthesis J Bacteriol 23 EF G I JK L M PQRT UV WgyrBR Database comparison: o PAL, no ß-oxidation enzymes! o prenyltransferase!
ounts (DPM) 14 - - Biosynthesis of prenylated benzoate moiety Biosynthesis of prenylated benzoate moiety 3 3 2 ovobiocin 3 l lorobiocin 2 l ovobiocin lorobiocin 3 3 3 3 3 3 Database comparison: 3 3 oumermycin A1 Dehydrogenase o sequence homology lass II aldolase E F G I JK L M PQRT UV WgyrB R E F G Y I JKLM 1 23 4 5 6 7 halp QR T U VW Z gyrb R pary R E G Y I JKLM P R RRR RR gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R pary R E F G I JK L M PQRT UV WgyrB R E F G Y I JKLM 1 23 4 5 6 7 halp QR T U VW Z gyrb R pary R E G Y I JKLM P R RRR RR gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R pary R Biosynthesis of prenylated benzoate moiety ypothesis 2 (hen & Walsh 21) Inactivation of Q 2 ov ov 2 ovi ov 2 2 A Wild-type (WT) I Bam PvuII probe Q (975 bp) B/P PvuII 1469 bp B Pojer et al. PA 1: 2316 (23) pfp4 WT Q8 Q mutant 3.6-kb 2.8-kb 1.9-kb Tyr Retro-aldol (a) ovf? ovr? ovq? R pfp4 Q - mutant B/P B/P III PvuII ind Bam I Pst I thio loq* (165 bp) PvuII I Bam 223 bp double cross-over recombination PvuII 2947 bp 1.5-kb robiocin wild-type Q - mutant Q - mutant + Q is involved in biosynthesis 696, 589, 58, 226 696, 589, 58, 226 696, 589, 58, 226 min. 23 37 Purification of loq and prenylation assays 3-dimethylallyl- 4-hydroxybenzaldehyde PRTEI: total soluble purified 97 kda 66 kda 45 kda 3 kda Tyr loq -GT 2 ov loq 2 ov ovi a 2 loq DMAPP loq ov DMAPP loq DMAPP (A) loq + DMAPP + [U- ] ß--Tyr--ov tyrosine 4-hydroxybenzaldehyde 3 1 3 ß-tyrosine 4.5 min. 17.3 min. Retention time (min.) RingA Pojer et al. PA 1: 2316 (23) 4 A Wild-type (WT) pew2 I - mutant indiii / / / coi Inactivation of I probe I (1221 bp) coi thio PstI coi coi loj 858 bp coi 1987bp XbaI double cross-over recombination loj coi loj loi* (12 bp) 858 bp B 1.9-kb.9-kb pew2 WT I4 loi mutant 34 nm 254 nm 696, 589, 58, 226 robiocin wild-type I - mutant 696, 589, 58, 226 D wild-type wild-type I - mutant 2 5, 1 61, 1 6 25, 1 61, 16 min. min. 23 37 14 25 I - mutant still produces prenylated 4-hydroxybenzoate Pojer et al. PA 1: 2316 (23)
ounts (DPM) ounts (DPM) Biosynthesis of prenylated benzoate moiety Q encodes a 4-hydroxyphenylpyruvate 3-dimethylallyltransferase ypothesis 2 hen & Walsh 21 Tyr?? 2 lo 2 lo loi Retro-aldol (a) lof? lor? 2 lo loj/k? loq? R I is not involved in biosynthesis 2 4PP k m= 25 µm 2 1 3 loq DMAPP k m= 35 µm loq + [1 14 ]DMAPP + 4PP Retention time (min.) 4 2 1 3 a loq + [1 14 ]DMAPP + 4PP; after a treatment 3-dimethylallyl- 4-hydroxyphenylpyruvic acid 24.92 min. min 3-dimethylallyl- 4-hydroxybenzaldehyde 26.35 min. 25.4 min Retention time (min.) 4 Pojer et al. PA 1: 2316 (23) loq represents a el prenyltransferase UbiA (ubiquinone) Lepgt 1 (shikonin) loq (robiocin) lor encodes a el bifunctional non-heme iron (II) dependent dioxygenase kda 1 2 3 4 97 66 45 3 lor-gt (56.5 kda) lor (3.5 kda).. 4PP.. 179, 135, 17 3DMA-4MA 235, 191, 189, 134 25, 161, 15, 15 3DMA-4PP 247, 23, 175 2 membrane-bound (D/)DxxD motif Mg 2+ dependent n soluble protein no (D/)DxxD motif Mg 2+ independent loq DMAPP lor lor + Fe 2+ + Fe 2+ Melzer et al. BBA 1994 Mühlenweg et al. Planta 1998 Pojer et al. PA 23 Kuzuyama et al. ature 25 4PP 3DMA-4PP 3DMA-4MA Pojer et al. JB 278: 3661 (23) Primary metabolism Biosynthesis of and Prephenate lof Tyr Transaminase 2 lo loq DMAPP 2 lo loi lor 2 2 lor 4PP E F G IJKL M PQRT UV WgyrB 2 lo loj/k? 2 R E F G Y IJKLM 1 23 4 5 6 7 halp QR T U VW Z gyrb R pary R 2 2 Linkage of rings 3 TDP ovv ovt ovw ovu ov ov ovi ovj ovk? ov ovf? ovq ovr Glucose Tyr Prephenate
Linkage of rings Linkage of rings Amide synthetase ovl ATP AMP Glycosyl transferase ovm Amide synthetase ovl ATP AMP 3 TDP ovl lol oul MW [kda] 94 67 43 3 3 TDP ovm: Thorson group: rg Lett 5: 933 (23) Walsh group: Biochemistry 42: 4179 (23) teffensky et al. JB 275: 21754 (2); Galm et al. hem & Biol 11: 173 (24) Tayloring reactions: ovobiocin Tayloring reactions: lorobiocin Methyltransferase ovp arbamoyl transferase ov AM 3 3 3 Xu et al. MGG 268: 387 (22) Xu et al. hem & Biol 11: 655-662 (24) P 2 3 Li et al. Microbiology 148: 3317 (22) Acyl transfer: APs lo1 & lo5 Transferases lo2 & lo7 Xu et al. hem & Biol 24 Freitag et al. hembiochem 25 Walsh group Biochemistry 26 3 Methyl transferase lo6 Westrich et al. hembiochem 4: 1 (23) 3 3 -lo5 Proline l lo5 Xu et al. MGG 268: 387 (22) lo4 lo3 Wang et al. AA 44: 34 (2) entral pyrrole unit of mermycin Functional analysis of biosynthetic gene clusters 3 l ovobiocin lorobiocin 2 3 3 3 3 3 3 3 3 oumermycin A1 EF G I JK L M PQRT UV WgyrB R E F G Y I JKLM 1 23 4 5 6 7 halp QR TUVWZ gyrb R pary R pary E G Y I JKLM P R RRR RR gyrb 1 23 4 5 67 1 2 3 4 5 6TUVW R R 3 2 3 Regulation E F G E G 3 E F G ovobiocin 3 3 oumermycin A1 3 l IJKL M P QR T UV W IJK L M 123 4 5 6 7 P R R R R 1 2 3 4 3 3 IJKLM 123 4 5 6 7 hal P QR T U VW lorobiocin R 6 TUVW
Functional analysis of biosynthetic gene clusters 3 3 2 ovobiocin 3 l lorobiocin Acknowledgements Pharmaceutical Biology Team, Tübingen Dr. B. Gust Prof..-M. Li 3 3 3 3 3 oumermycin A1 3 E F G IJKL M P QR T UV WgyrB R Resistance Regulation E F G Y IJKLM 123 4 5 6 7 hal P QR T U VW ZgyrB R pary R E G Y IJK L M 123 4 5 6 7 P R R R R 1 2 3 4 R R gyrb pary 6 TUVW R R 5 ooperation partners L. Wessjohann, alle A. Maxwell, JI orwich, UK G. Bringmann, Würzburg Basilea AG, witzerland B. Kammerer, Tübingen Funding Deutsche Forschungsgemeinschaft European ommission