large family of structurally diverse and natural products produced by bacteria and fungi
DESCRIPTION
3.5kdb. Ladder 0h 1h 2h 4h 6h 8h 10h 24h. Isolation & Characterization of Polyketide Synthase Thioesth er ase Domains Evelyn Walenta & Christopher N. Boddy* Department of Chemistry, Syracuse University Sy racuse, NY 13244. - PowerPoint PPT PresentationTRANSCRIPT
Isolation & Characterization of Polyketide Synthase Thioestherase Domains
Evelyn Walenta & Christopher N. Boddy*Department of Chemistry, Syracuse University
Syracuse, NY 13244
O OO
O
O
O
O
H
H
OO
O
OH
O
O
O
O
O OH OH
OH
OH OH
OH
O
OH
COONaHO
O OHNH2
OH
O
O
O
OH OH OH
OH
OH
OH O
OH
COOHHO
O OHNH2
OH
O
large family of structurally diverse and natural products produced by bacteria and fungi
Polyketides are important clinical agents
Polyketides are made in an assembly-line fashion
Project Goal: Characterize multiple TE Domains
Generation of TE expression vector
~950
Amphotericin TE Protein Expression
~6.5 ~5.5Ladder 0h 1h 2h 4h 6h 8h 10h 24h
3.5kdb
Activity of Amphotericin TE
Acknowledgements
Ben Lundgren (Syracuse University)
Mike Henry (Syracuse University)
The Boddy Lab (Syracuse University)
Syracuse University
University of Technology (Graz)
Amphotericin Nystatin
Avermctin Oleandomycin
O
O
OAc
O O
O
O
O
O
OAc
N
OCH3
OAc
Release of Polyketide from enzyme in crucial
Thioester domain catalyzes the release of the polyketide via cyclization
Enormous multifunktional enzymes
Growing polyketide is covalently attached to enzyme
Complex mulecules are made from simple starting materials
Different organisation of modules gives different polyketides
- HSR
R: ACP or
N-Acetulcysteamine
cyclorelease
Amphotericine, Nystatin, Avermectin, Oleandomycin TE domains were selected
TE domains were sequenced
bacterial strains were commercially available
Polyketide products were commercially available
Why?
producing organisms
grow culture
isolate genomic DNA
PCR
PCR products for all 4 TE domains were generated
BL21 (CDE3) were used for protein expression)
Time course for protein expression at 30°C shows 10h to be optimal
Amphotericin TE was purified by Ni-NTA chromatography
4 ml of 22 µM high purity Amphotericin TE was obtained from 0.5 l of culture
Amphotericin TE does not catalyze lactone hydrolosys
O
O OH OH
OH
OH OH
OH
O
OH
COONaHO
O OHNH2
OH
O
O OH OH
OH
OH OH
OH
O
OH
COONaHO
O OHNH2
OH
O
HOHO
TE domain
LCMS analysis shows no hydrolysis activity
Thioester Domain can hydrolyze simple Thioesters
NS
HAc
O
NSH
HAc
HO
OTE domainExpression vector for
Amphotericin TE was generated
clone into expression vecoor
LCMS analysis shows hydrolysis activity