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Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS.
- Source :
-
Chemistry & biology [Chem Biol] 2011 Sep 23; Vol. 18 (9), pp. 1075-81. - Publication Year :
- 2011
-
Abstract
- Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.<br /> (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Subjects :
- Acyl Coenzyme A metabolism
Anti-Bacterial Agents chemistry
Catalytic Domain
Hydrolysis
Kinetics
Macrolides chemistry
Mutation
Polyketide Synthases genetics
Polyketide Synthases metabolism
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Substrate Specificity
Acyl Coenzyme A chemistry
Anti-Bacterial Agents biosynthesis
Fourier Analysis
Macrolides metabolism
Mass Spectrometry
Polyketide Synthases chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1879-1301
- Volume :
- 18
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Chemistry & biology
- Publication Type :
- Academic Journal
- Accession number :
- 21944746
- Full Text :
- https://doi.org/10.1016/j.chembiol.2011.07.016