title: Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster.
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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Aron
authors.forename: Pieter C
authors.initials: PC
authors.lastname: Dorrestein
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Blackhall
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D009097:Multienzyme Complexes; C012448:mycosubtiline; D048630:Polyketide Synthases; D000637:Transaminases