Probing intra- versus interchain kinetic preferences of L-Thr acylation on dimeric VibF with mass spectrometry.
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.
Hicks
Leslie M
LM
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Balibar
Carl J
CJ
Walsh
Christopher T
CT
Kelleher
Neil L
NL
Hillson
Nathan J
NJ
D000215:Acylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry*
D001426:Bacterial Proteins; D013912:Threonine; D010453:Peptide Synthases; C405545:VibF protein, bacteria