HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, L. M. Articles by Kelleher, N. L. Search for Related Content PubMed PubMed Citation Articles by Miller, L. M. Articles by Kelleher, N. L. Social Bookmarking                   What's this?

Parallel interrogation of covalent intermediates in the biosynthesis of gramicidin S using high-resolution mass spectrometry

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

(RECEIVED April 29, 2005; FINAL REVISION July 12, 2005; ACCEPTED July 20, 2005)

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several nonnatural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of noncognate dipeptides on the second module.

Keywords: Fourier-transform mass spectrometry; nonribosomal peptide synthesis; gramicidin S; covalent intermediates

Abbreviations: MS, mass spectrometry • Q-FTMS, quadrupole-Fourier-transform mass spectrometry • TEII, type II thioesterase • NRPS, nonribosomal peptide synthases • PKS, polyketide synthases • GrsA, gramicidin S synthetase I • GrsB, gramicidin S synthetase II • A, adenylation domain • T, thiolation domain • E, epimerization domain • C, condensation domain • TE, thioesterase domain • DKP, diketopiperazine • CNBr, cyanogen bromide • CoA, coenzyme A • TCEP, tris(2-carboxyethyl)phosphine hydrochloride • RPLC, reverse-phase liquid chromatography • ESI, electrospray ionization • M_r, relative molecular weight • MS/MS, tandem MS • m, change in mass • PTM, post-translational modification • PCP, peptidyl carrier protein.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051553705.

Reprint requests to: Neil L. Kelleher, Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA; e-mail: kelleher{at}scs.uiuc.edu; fax: (217) 244-8068.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. M. Hicks, C. J. Balibar, C. T. Walsh, N. L. Kelleher, and N. J. Hillson Probing Intra- versus Interchain Kinetic Preferences of L-Thr Acylation on Dimeric VibF with Mass Spectrometry Biophys. J., October 1, 2006; 91(7): 2609 - 2619. [Abstract] [Full Text] [PDF]