The Career of Christopher T. Walsh

Supergroup Meeting May 9, 2007

Teresa D. Beeson

Outline

! Who is Christopher T. Walsh?

! Research

! Suicide Substrates

! Vancomycin Resistance

! Siderophores

! Biosynthesis of Natural Products

1 Biography

Christopher T. Walsh

Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

Biography

. 1965 A.B. in biology (Harvard Univ.)

. C. Walsh, J.H. Law, and E.O. Wilson. "Purification of the Fire Ant Trail Substance." Nature 1965, 207, 320-321.

. 1970 Ph.D. in life sciences (Rockefeller Univ.)

. Advisor Leonard B. Spector

. 6 publications, all first author

. "The Mechanism of Action of the Citrate Cleavage Enzyme." Ph.D. Dissertation, The Rockefeller University.

2 Biography

. 1970-1972 Postdoctoral Fellow (Brandeis Univ.)

. Advisor Robert H. Abeles

. 8 publications

. 1972-1987 Professor of Chemistry and Biology (MIT)

. 1987-1995 Chair, Dept. of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

. 1991-present Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

. 1992-1995 President of Dana Farber Cancer Institute

Biography

. Authored over 650 publications since 1965

. Average of over 15 publicatons per year for 42 years

. Former students and post docs include:

. Michael Marletta (Berkeley) . Robert Pascal (Princeton) . Peter Schultz (Scripps) . Greg Verdine (Harvard) . Yian Shi (Colorado State) . Thomas Wandless (Stanford)

3 Affiliations

. National Academy of Sciences

. Institute of Medicine

. American Academy of Arts and Sciences.

. American Academy of Microbiology

. Pharmaceutical consultant (Merck, Roche, and Abbot)

. Scientific advisor (Microbia, Genzyme, Immunogen, Kosan Biosciences, and Millennium)

. Board of Directors (Critical Therapeutics, Kosan, Immunogen, Leukosite. Microbia, Transform, and Vicuron)

Awards

. Eli Lilly Award in Biochemistry

. Arthur C. Cope Scholar Award in Organic Chemistry

. Repligen Award in Biological Chemistry

. Alfred Bader Award in Bioorganic and Bioinorganic Chemistry

. Promega Biotechnology Research Award from the American Society for Microbiology (ASM).

4 Suicide Substrates

Suicide Substrates

! What is a suicide substrate?

! A competitive inhibitor that is converted to an irreversible inhibitor at the active site of the enzyme

O O O O HO HN Me N Me N Me HN Me

NADPH, O2 – H2O

P450 P450

OH OH O OH

Acetaminophen Imino quinone Alkylated Enzyme Tylenol

! The enzyme causes it's own destruction by unmasking a latent functional group

Walsh, C. T. Tetrahedron, 1982, 38, 871.

5 Suicide Substrates

! Suicide substrates in medicine

! Chemotherapy Me O

O O Me H F HN HO NH2 H H NH2 O O N F F H OH 5-Fluorouracil DFMO Formestane

! Antibiotics O OH NH2 O N O N O S H O O Sulbactam D-cycloserine

! Suicide substrates for research

! Can be used study the role of proteins in vitro or in vivo

Walsh, C. T. Tetrahedron, 1982, 38, 871.

Suicide Substrates

! Enzyme Target: E. coli B Alanine Racemase

! Alanine racemase epimerizes L-alanine

O O O Me alanine racemase Me Me OH OH OH

NH2 NH2 NH2

L-alanine D-alanine

! D-Alanine incorporated into bacterial cell walls ! D-Ala-D-Ala binding site for Vancomycin ! Racemases not found in mammalian cells

! Natural Antibiotics for Alanine Racemase

NH2 O O

O H2N O OH N O H NH2

D-cycloserine O-carbamoyl-D-serine

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

6 Suicide Substrates

! Haloalanines also shown to be bacteriocidal

! Time-dependant inhibition of alanine racemase

O O

Cl OH F OH

NH2 NH2

D-!-chloroalanine D-!-fluoroalanine

! Deuterated analogues more potent antibiotics

D D O D D O

Cl OH F OH

NH2 NH2

! What is the mode of enzyme deactivation?

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

Suicide Substrates

! Observations from the Walsh Lab

! Time-dependant inactivation of alanine racemase by fluoroalanine

! Enzyme activity was never regained after filtration or competitive binding ! 14C-Labeled fluoroalanine caused 14C-labeling of enzyme ! Haloalanines deemed to be irreversible inhibitors

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

7 Suicide Substrates

! Observations from the Walsh Lab

! Alanine racemase did not epimerize haloalanines

O O O Me alanine racemase Me Me OH OH OH

NH2 NH2 NH2

L-alanine D-alanine

O O

alanine racemase Me – Cl OH OH NH3 Cl

NH2 O

Pyruvate

! D-haloalanines much faster than L-haloalanines but...

! Turnovers before inactivation ~800 for all haloalanines

! Is a common inactivator or "suicide substrate" formed?

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

Suicide Substrates

! Explaining the observations

O O

– Cl O x O–

NH3 NH3 Alkylated Enzyme

! Simple nucleophilic attack by enzyme we would expect less turnovers before inactivation for Cl than F

! Haloalanines must be suicide substrates

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

8 Suicide Substrates

! Explaining the observations O H Cl O– H O O H N – O O O H P OH –O O Cl O– P OH HO O NH3 HO Me N Me N H H Pyridoxal-P Aldimine complex

O O

– Cl O– O H N H N O H –H+ O H – – –O – Cl O P OH P OH O O HO HO

Me N Me N H H Enamino acid complex Electrophilic species

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

Suicide Substrates

! Explaining the observations

O

O– H N O O H – O O H2O H2O Me P OH O– O– O HO NH3 O Me N Pyruvate H

Electrophilic species

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

9 Suicide Substrates

! Explaining the observations

O

O– H N O O H – O O H2O H2O Me P OH O– O– O HO NH3 O Me N Pyruvate H

Electrophilic species

O

O– H N H O – O O H2O P OH O O– HO O Me N H Alkylated Enzyme

Enzyme nucleophilic attack

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

Suicide Substrates

! Natural D-serine antibiotics determined to be suicide substrates

NH2 O O O O

O H2N O OH Me O OH N O H NH2 NH2

D-cycloserine O-carbamoyl-D-serine O-acetyl-D-serine

! Turnovers per inactivation also ~800

! D-serine does not inactivate alanine racemase

O

HO OH

NH2

D-serine

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

10 Suicide Substrates

! Propargyl suicide substrates operate in a similar manner

O O O

O– • O– O– H N H N NH O H O H O –O –O –O P OH P OH P OH O O O HO HO HO

N Me N Me N Me H H H

O H+ O O– O O– Me N O H Me N O– –O O H P OH –O Me NH O P OH 3 HO O HO N Me N Me H H

Johnston, M.; Jankowski, D.; Marcotte, P.; Tanaka, H.; Esaki, N.; Soda, K.; Walsh, C. T. J. Am. Chem. Soc., 1979, 21, 4690.

Suicide Substrates

! Allyl sulfoxide suicide substrates undergo 2,3-rearrangement

O O S O R O O – O S O S O– R Cl N O– H !-Elim R N H N OH H – OH HO3PO – HO3PO OH – HO3PO N Me N Me N Me H H H

sulfenate ester

OH O SR O– O

Johnston, M.; Raines, R.; Walsh, C.; Firestone, R. A. J. Am. Chem. Soc., 1980, 4241.

11 Vancomycin Resistance

Vancomycin Resistance

! Background: Vancomycin's mode of Action

Disaccharide pentapeptide units are cross-linked to build rigid bacterial cell walls Vancomycin-PG-D-Ala-D-Ala complex sterically prevents further cell wall assemply

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

12 Vancomycin Resistance

! The Key Missing H-Bond

! Key H-bond lost due to replacement of D-Alanine for D-Lactate

! 1000-fold reduction in Kd

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

Vancomycin Resistance

! 5 Genes Necessary for Vancomycin Resistance

P vanR vanS vanH vanA vanX vanR PvanH

! All 5 encoded proteins isolated and characterized by Walsh Lab

Protein Activity Function

VanR Transmembrane histidine kinase Initiates signal transduction

VanS Response regulator Activates vanH,A,X transcription

VanH D-specific !-keto acid reductase Generates D-lactate

VanA Depsipeptide ligase for D-Ala-D-lactate Generates D-Ala-D-lactate

VanX D-Ala-D-Ala dipeptidase Removes D-Ala-D-Ala

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

13 Vancomycin Resistance

! VanR and VanS: Enteroccocal Bacteria's "Sensor"

In the absence of Vancomycin, VanH,A,X genes are not transcribed VanS located in the bacterial cell wall, phosphorylated upon external signal

Phosphate transferred to VanR, which causes conformational change

VanR-PO3 is DNA-binder to promoter region of vanH,A,X, induces transcription

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

Vancomycin Resistance

! Key Change from Alanine to Lactate

O OH O VanH O Me Me O O Pyruvate D-Lactate

OH Me O Me O H O VanA O N Me H2N O H2N O O O Me O Me D-Lactate D-Ala-D-Lactate D-Ala-D-Ala

Me O O H N VanX 2 H2N H2N O O O Me Me D-Ala-D-Ala 2 D-Ala

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

14 Vancomycin Resistance

! The Key Missing H-Bond

! Key H-bond lost due to replacement of D-Alanine for D-Lactate

! 1000-fold reduction in Kd

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

Siderophores

15 Siderophores

! “Bacterial-Host Iron Wars”

! Iron required for bacterial growth

! Concentration of Fe(III) in water 10–18 M

! Concentration of Fe(III) in human serum ~10–24 M

! Bacterial growth requires cytoplasmic Fe(III) concentration of ~10–6 M

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

Siderophores

! Bacteria secrete "siderophores" to scavange iron

! "Sidero" means iron; "phore" means to carry in greek

! Low molecular weight molecules with high Fe(III) affinity

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

16 Siderophores

! Common siderophores

OH O OH O HO OH O OH H N NH OH O HN NH O O O O O H O O O S OH HO OH OH S N O N O H S N HN O Me OH N OH HO N HO O O Yersiniabactin Me Y. pestis Me HO "plague" Aerobactin Enterobactin E. coli E. coli O Salmonella

N OH OH O O H N O N OH N N H O O O Me Mycobactin J M. paratuberculosis "tuberculosis"

Siderophores

! Siderophores studied by the Walsh Lab

! Enterobactin from E. coli and salmonella

! Mycobactin from Mycobacterium tuberculosis

! Pyochelin from Pseudomonas aeruginosa

! Yersiniabactin from Yersinia pestis "plague"

! Vibriobactin from Vibrio cholerae "cholera"

17 Siderophores

! Enterobactin-mediated iron aquisition

OH OH O O OH H N NH OH O O O O O

O HN O

HO

HO

Enterobactin

–49 Fe(III) Kd = 10 M

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

Siderophores

! Enterobactin-mediated iron aquisition

OH OH O O OH H N NH OH O O O O O

O HN O

HO

HO

Enterobactin

–49 Fe(III) Kd = 10 M

"It can rip iron out of stainless steel" – C. T. Walsh

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

18 Siderophores

! Production of iron scavangers alone not sufficient

! Iron-free form must be secreted through bacterial cell wall

! Fe(III) scavanged from surrounding environment

! Iron-bound form imported through bacterial cell wall

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

Siderophores

! E. coli turn on 15 genes when iron deficient

! EntA-F: Synthesis of enterobactin from chorismate and serine

! EntS: Export out of cell wall

! FepA-G: Import of iron-bound enterobactin

! Fes: Release of Fe(III) from enterobactin

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

19 Siderophores

! Immune system's response to enterobactin

! Serum albumin binds enterobactin

! Siderocalin, from neutrophils, binds enterobactin

! Enterobactin sequestered and rendered ineffective

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

Siderophores

! "Bacteria fight back"

! iroA: 5 gene cluster, encodes 5 proteins

! IroB and IroE: modification of enterobactin before export

! IroC: Export of modified enterobactin

! IroN: Import of iron-bound modified-enterobactin

! IroD: Release of iron from complex

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

20 Siderophores

! "Bacteria fight back"

! IroB C-glycosylation of enterobaction

! Biologically very rare, extremely stable

OH OH O OH OH O OH O H OH O HO N NH OH O OH HO O H OH N NH OH O O O O O O O O O O IroB HN O O HN O HO

HO HO OH O HO HO OH OH

Enterobactin Salmochelin S4 (DGE)

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

Siderophores

! "Bacteria fight back"

! IroE linearization of Salmochelin followed by IroC-mediated export ! Salmochelin S2 30-fold lower lipid partition coefficient than enterobactin

OH OH O OH OH O OH O H OH O HO N NH OH OH HO O OH O O H OH HO N NH OH O HO O O O O OH O O HO O O HN O IroE O OH HN O HO HO HO OH O HO OH O HO OH OH HO OH OH Salmochelin S4 (DGE) Salmochelin S2

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

21 Siderophores

! "Bacteria fight back"

! IroD exhaustively hydrolyzes iron-bound Salmochelins

OH OH OH O O O O H O 2 HO N HO OH O O OH OH HO O HN O OH O NH HN O Salmochelin SX O O O IroD

OH O HO O O OH O O H N HO OH OH HO O OH Iron-bound Salmochelin S2

Fe(III)

! Steric bulk and hydrophilic nature of glucoses prevent siderocalin binding

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

22 Natural Product Biosynthesis

Nature's Assembly Lines

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468.

23 Nature's Assembly Lines

! Many biologically-active natural products fall into 3 classes

! Non-ribosomal peptides (NRP)

! Building blocks of amino acids and aryl acids

! Polyketides (PK)

! Comprised mainly of malonyl-CoA, methylmalonyl-CoA

O O O O CoA CoA HO S HO S Me NH2 OH N H H O O N N N s-CoA = S O P O P O N N O O O– O– O

–O P O OH O– ! Hybrids of polyketides and non-ribosomal peptides

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468.

Natural Product Assembly Lines

! Yersiniabactin is a NRP-PK hybrid siderophore

Me S OH HO H N N S O S H N

HO

Yersiniabactin

! Yersiniabactin has been synthesized in 35 chemical steps

! Enzymolic assembly line uses 15 steps

Ino, A.; Murabayashi, A. Tetrahedron, 2001, 57, 1897.

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

24 Natural Product Assembly Lines

! Functional "domains" within proteins form assembly line

T thiolation Cy cyclization

KS ketosynthase CE condensation/epimerization

AT acyltransferase E epimerization

C condensation MT methyltransferase

CLF chain length factor Re reductase

A adenylation Ox oxidase

TE thioesterase AL acyl-CoA ligase

ER Enoylreductase AMT aminotransferase

DH dehydratase Hal halogenase

KR ketoreductase Cyp cyclopropanase

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468.

Natural Product Assembly Lines

! Acylation of "T" thiolation domain starts NRP and PK assembly

A A Cy ~ A Cy A T T ~ T T OH B SH S– OH O NH2 NH2 N N O O O N O N –O P O P O P O O P O N N N N O– O– O– O O– O OH O

ATP OH OH OH OH

A Cy A ~ T T

S O

HO

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

25 Natural Product Assembly Lines

! Cyclization "Cy" domains catalyze thiazoline/oxazoline formation

A Cy A ~ A Cy A ~ T T T T B B S S S O H O O N HO H HS NH HS O

HO

A Cy A ~ A Cy A ~ T T T T B B S S O O

S N S NH HO OH OH

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

Natural Product Assembly Lines

! Additional cyclization "Cy" domains & tandem heterocyclizations

T ~ T ~ A Cy A Cy A Cy A Cy T T T S T S B O B O

S H2N O S N HS N S N HO HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

26 Natural Product Assembly Lines

! PK ketosynthase "Ks" domains catalyze C–C bond formation

AT AT KS AT KS MT T KS MT ~ MT ~ T T T ~ T T OH O SH OH AcS SH AcS O S S S– O O O O O Me –O SCoA O– O O–

AT AT KS MT KS MT T T ~ T T ~ S SH O S S Me O O– O Me

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

Natural Product Assembly Lines

! PK ketosynthase "Ks" domain also catalyzes chain transfer

NRP Assembly Line PK Assembly Line Hybrid NRP-PK ~ T KS KR A Cy A Cy KS AT AT KR MT T ~ T T MT ~ S T S O SH O S S O S N O S N O O – N N O O– HO S HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

27 Natural Product Assembly Lines

! Methyltransferase "MT" domains transfer Me from SAM-Me

KS KR KS AT 2 Me-SAM AT KR MT T ~ MT T ~

S S O O

OH O

S N S N

N N HO S HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

Natural Product Assembly Lines

! Ketoreductase "KR" domains reduce ketones with NADPH

KS KR KS AT NADPH AT KR MT T ~ MT T ~

S S O O N

O NH2 OH H H S N O S N

N N HO S HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

28 Natural Product Assembly Lines

! Cyclization then methylation yields quaternary stereocenter

MT KS KR T Cyclization MT AT Cy KS KR T MT T TE AT Cy TE Methylation MT T S S S HS O O Me O NH2 N OH S

S N OH

S N N HO S N HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

Natural Product Assembly Lines

! Terminal thioesterase "TE" hydrolyzes ester linkage

KS MT AT KR Cy T MT T TE YbtU Me S OH HO H S N N HO post-translational S O O reduction Me S H N N S HO

OH Yersiniabactin

S N

N HO S

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

29 Natural Product Assembly Lines

! How big are the assembly lines?

! Most are >1 MDa or 10,000 amino acids ! Tyrocidine synthetase has 30 domains in 3 proteins, ~1 MDa

! Largest known is PK ECO-02301

! 122 domains ! 9 proteins ! 4.7 MDa, 47,000 amino acid residues

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468. Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.

Macrocyclization of Natural Products

! Macrocycles Constrain, Induce Bioactivity

! Structures of cyclic natural products from NRPS (surfactin, daptomycin, iturin, ramoplanin, and tyrocidine) and PKS (erythromycin) systems.

Tseng, C. C.; Bruner, S. D.; Kohli, R. H.; Marahiel, M. A.; Walsh, C. T.; Sieber, S. A. Biochem, 2002, 41, 13350.

30 Macrocyclization of Antibiotics

! Thioesterase Identified as Catalyst for Macrocyclization

! HPLC analysis shows disappearance of pentapeptide and appearance of Tyrocidine A in the presence of TyrC Thioesterase

Trauger, J. W.; Kohli, R. H.; Mootz, H. D.; Marahiel, M. A.; Walsh, C. T. Nature, 2000, 407, 215.

Macrocyclization of Antibiotics ! Tyrocidine Thioesterase Catalyzes Macrocyclization

a) Tyrocidine non-ribosomal peptide synthetase b) Proposed mechanism and experimental system used as a probe

Trauger, J. W.; Kohli, R. H.; Mootz, H. D.; Marahiel, M. A.; Walsh, C. T. Nature, 2000, 407, 215.

31 Macrocyclization of Antibiotics

! Thioesterase Proposed to Catalyze Gramicidin Formation

! Proposed thioesterase catalyzed dimerization and macrocyclization

Trauger, J. W.; Kohli, R. H.; Mootz, H. D.; Marahiel, M. A.; Walsh, C. T. Nature, 2000, 407, 215.

Macrocyclization of Antibiotics

! Thioesterase Catalyzes Macrolactonization to Epothilone C

Boddy, C. N.; Schneider, T. L.; Hotta, K.; Walsh, C. T.; Khosla, C. J. Am. Chem. Soc., 2000, 125, 3428.

32 What Now?

Combinatorial Biosynthesis

! Solid-phase macrocyclization using thioesterase

PEGA O O

Leu–Orn–Val–Tyr–Gln–Asn–DPhe–Phe–Pro–DPhe-NH2

Isolated thioesterase domain

TE

Tyrocidine A

Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature, 2002, 418, 658.

33 Combinatorial Biosynthesis

! D-Phe replaced by natural and unnatural amino acids

PEGA O O

Leu–Orn–Val–Tyr–Gln–Asn–XXX–Phe–Pro–XXX-NH2 ! Library of over 300 new antibiotics synthesized

Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature, 2002, 418, 658.

Combinatorial Biosynthesis

! New antibiotics show different activity spectrum

Tyrocidine A !A02 !F10

Eukaryotic (MHC, µM) Human erythrocytes 4 60 150

Gram negative (MIC, µM) E. coli >290 120 290 P. aeruginosa 290 30 37 P. putida >290 30 74

Gram positive (MIC, µM) B. subtilis 1 2 5 MRSA 2 7 18 S. epidermidis 2 4 9

! Higher selectivity for baterial over human cells

Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature, 2002, 418, 658.

34 Summary

! Chris Walsh's publication record puts us all to shame

! Research encompasses vast number of enzymes

! "Trojan Horse" suicide substrates

! The 5 genes of vancomycin resistance

! "Bacterial-Host iron wars" Siderophores

! Assembly-Line enzymology of natural products

35