Probing intra- versus interchain kinetic preferences of L-Thr acylation on dimeric VibF with mass spectrometry

Leslie M. Hicks, Carl J. Balibar, Christopher T. Walsh, Neil L. Kelleher, Nathan J. Hillson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

We present a method to probe intra- and interchain activities within dimeric nonribosomal peptide synthetases. Utilizing domain inactivation and analytical mass mutants in conjunction with rapid-quench, mass spectrometry, and a probabilistic kinetic model, we have elucidated the pre-steady-state intra- and interchain rates and the corresponding flux of the acylation of L-Thr onto VibF. Although the intra rate is significantly faster than the inter rate, the data are most consistent with an even flux of covalent substrate loading where neither pathway dominates. These pre-steady-state results confirm previous steady-state in vitro mutant complementation studies of VibF. Extension of this methodology to other dimeric nonribosomal peptide synthetases, and to the related fatty acid and polyketide synthases, will further our biophysical understanding of their acyl-intermediate-processing pathways.

Original languageEnglish (US)
Pages (from-to)2609-2619
Number of pages11
JournalBiophysical Journal
Volume91
Issue number7
DOIs
StatePublished - Oct 2006

Funding

This work was supported by grants from the National Institutes of Health (GM 067725 to N.L.K., GM 20011 to C.T.W). N.J.H. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1880-05). We further acknowledge generous support from the Sloan Foundation, a National Science Foundation Graduate Research Fellowship to L.M.H., a Pharmacological Sciences Training Grant from the National Institute of General Medical Sciences, and an National Defense Science and Engineering Graduate Fellowship to C.J.B. We thank Michael Boyne and Frédéric Vaillancourt for reading of the manuscript, and Furong Sun and Richard Milberg for assistance in collecting data with the Q-TOF Ultima mass spectrometer, which was purchased in part with a grant from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085).

ASJC Scopus subject areas

  • Biophysics

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