12756
Biochemistry 2006, 45, 12756-12766
Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains via Phosphopantetheinyl Elimination Reactions during Tandem Mass Spectrometry† Pieter C. Dorrestein,‡,§ Stefanie B. Bumpus,‡ Christopher T. Calderone,| Sylvie Garneau-Tsodikova,|,⊥ Zachary D. Aron,| Paul D. Straight,@ Roberto Kolter,@ Christopher T. Walsh,| and Neil L. Kelleher*,‡ Department of Chemistry, UniVersity of Illinois, 600 South Mathews AVenue, Urbana, Illinois 61801, Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, Boston, Massachusetts 02115, and Department of Microbiology and Molecular Genetics, HarVard Medical School, Boston, Massachusetts 02115 ReceiVed June 12, 2006; ReVised Manuscript ReceiVed July 28, 2006
ABSTRACT: With the emergence of drug resistance and the genomic revolution, there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultralarge enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gasphase carrier domains, facile elimination occurs in benchtop and Fourier-transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to
