TAILIEUCHUNG - Báo cáo khoa học: The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry

A catalytically competent, homodimeric diketide synthase comprising the first extension module of the erythromycin polyketide synthase was analysed using MS, after limited proteolysis to release functional domains, to deter-mine the pattern of covalent attachment of substrates and intermediates to active sites during catalysis. | The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry Hui Hong1 2 Peter F. Leadlay2 and James Staunton1 1 Department of Chemistry University of Cambridge UK 2 Department of Biochemistry University of Cambridge UK Keywords enzyme-bound intermediate erythromycin limited proteolysis liquid chromatographymass spectrometry polyketide synthase Correspondence H. Hong Department of Biochemistry University of Cambridge 80 Tennis Court Road Cambridge CB2 1GA UK Fax 44 1223 766002 Tel 44 1223 333659 TE-mail hh230@ J. Staunton Department of Chemistry University of Cambridge Lensfield Road Cambridge CB2 1EW UK Fax 44 1223 336362 Tel 44 1223 336300 E-mail js24@ Received 28 July 2009 revised 29 September 2009 accepted 30 September 2009 doi A catalytically competent homodimeric diketide synthase comprising the first extension module of the erythromycin polyketide synthase was analysed using MS after limited proteolysis to release functional domains to determine the pattern of covalent attachment of substrates and intermediates to active sites during catalysis. Using the natural substrates the acyltransferase and acylcarrier protein of the loading module were found to be heavily loaded with propionyl starter groups while the ketosynthase was fully prop-ionylated. The acylcarrier protein of the extension module was partly occupied by the product diketide and the adjacent chain-releasing thioesterase domain was vacant implying that the rate- limiting step is transfer of the diketide from the acylcarrier protein to the thioesterase domain. The data suggest an attractive model for preventing iterative chain extension by efficient repriming of the ketosynthase domain after condensation. Use of the alternative starter unit valeryl-CoA produced an altered pattern in which a significant proportion of the extension acylcarrier protein was

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