Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster |
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Authors: | Aron Zachary D Dorrestein Pieter C Blackhall Jonathan R Kelleher Neil L Walsh Christopher T |
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Institution: | Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. |
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Abstract: | We report the expression and characterization of a truncated form of MycA from the Mycosubtilin gene cluster from Bacillus subtilis. The MycA fragment contains a new amino transferase (AMT) tailoring domain, allowing the first detailed study of a PLP-dependent enzyme operating in cis within the PKS and NRPS biosynthetic paradigm. As the AMT domain acts on covalently bound beta-ketothioesters, and is therefore a single-turnover system, electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS) was used to observe the amine-transfer reaction both for amine donor substrate specificity and to regiospecifically determine enzyme-bound intermediates. We confirm the function of the AMT domain, dissect the mechanistic steps of amine transfer, identify the preferred amine source, and localize the beta-ketothioester substrate during amine transfer. |
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