tailieunhanh - Báo cáo khoa học: Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry

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 exten-sion module linked to TE (diketide synthase, DKS). | ềFEBS Journal Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry Hui Hong1 Antony N. Appleyard2 Alexandros P. Siskos2 Jose Garcia-Bernardo2 James Staunton1 and Peter F. Leadlay2 1 Department of Chemistry University of Cambridge UK 2 Department of Biochemistry University of Cambridge UK Keywords erythromycin limited proteolysis liquid chromatography-mass spectrometry multienzyme polyketide synthase Correspondence J. Staunton Department of Chemistry University of Cambridge Lensfield Road Cambridge CB2 1EW UK Fax 44 1223 762018 Tel 44 1223 766041 E-mail js24@ Received 10 November 2004 revised 28 January 2005 accepted 15 February 2005 doi 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 or n-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 .