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- Title
Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry.
- Authors
Hui Hong; Appleyard, Antony N.; Siskos, Alexandros P.; Garcia-Bernardo, Jose; Staunton, James; Leadlay, Peter F.
- Abstract
Limited proteolysis in combination with liquid chromatography-ion trap mass spectrometry (LC-MS) was used to analyze engineered or natural proteins derived from a type I modular polyketide synthase (PKS), the 6-deoxyerythronolide B synthase (DEBS), and comprising either the first two extension modules linked to the chain-terminating thioesterase (TE) (DEBS1-TE); or the last two extension modules (DEBS3) or the first extension module linked to TE (diketide synthase, DKS). Functional domains were released by controlled proteolysis, and the exact boundaries of released domains were obtained through mass spectrometry and N-terminal sequencing analysis. The acyltransferase-acyl carrier protein required for chain initiation (ATL-ACPL), was released as a didomain from both DEBS1-TE and DKS, as well as the off-loading TE as a didomain with the adjacent ACP. Mass spectrometry was used successfully to monitor in detail both the release of individual domains, and the patterns of acylation of both intact and digested DKS when either propionyl-CoA orn-butyryl-CoA were used as initiation substrates. In particular, both loading domains and the ketosynthase domain of the first extension module (KS1) were directly observed to be simultaneously primed. The widely available and simple MS methodology used here offers a convenient approach to the proteolytic mapping of PKS multienzymes and to the direct monitoring of enzyme-bound intermediates.
- Subjects
PROTEIN metabolism; PROTEOLYSIS; PROTEINS; PROTEIN binding; MASS spectrometry; POLYMERS
- Publication
FEBS Journal, 2005, Vol 272, Issue 10, p2373
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2005.04615.x