N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF

Abstract

Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr(1) amino group and generate the fatty acyl-Thr(1)-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis.

Figure 1

Chemical Structures of Glidobactin A and Syringolin A. See also Figure S1

Figure 2

Characterization of the Glidobactin Initiation Module, GlbF. A) Partial glidobactin cluster. B) GlbF module organization and putative thioester product. C) Phylogenetic analysis of C domains including the GlbF starter C domain. See also Figures S1, S2

Figure 3

HR LC-MS spectra of condensation products hydrolyzed from the GlbF T–domain. A) 2(E),4(E) dodecadienoyl-Thr Product. B) 2(E),4(E) decadienoyl-Thr Product. See also Figures S3, S4, and S5.