-
[show abstract]
[hide abstract]
ABSTRACT: The recognition of carrier proteins by multiple catalytic partners occurs in every cycle of chain elongation in the biosynthesis of fatty acids and of the pharmacologically important polyketide and nonribosomal peptide natural products. To dissect the features of carrier proteins that determine specific recognition at distinct points in assembly lines, we have used the two-module Escherichia coli enterobactin synthetase as a model system. Using an entB knockout strain, we developed a selection for growth on iron-limiting medium to evolve aryl carrier protein domains. The aryl carrier proteins from VibB of Vibrio cholerae vibriobactin and HMWP2 of Yersinia pestis yersiniabactin assembly lines were evolved by random mutagenesis to support growth under selection conditions, yielding a convergent set of mutations. Subsequent in vitro biochemical characterizations with partner enzymes EntE, EntF, and Sfp on the evolved VibB aryl carrier protein revealed a approximately 500-fold improvement in reconstituted enterobactin production activity. Mechanistic characterization identified three distinct specific recognition surfaces of VibBArCP for three catalytic partners in enterobactin biosynthesis. Our results suggest that heterologous carrier protein interactions can be engineered with a small number of mutations given a suitable selection scheme and provide insights for reprogramming nonribosomal peptide biosynthesis.
Proceedings of the National Academy of Sciences 08/2007; 104(28):11621-6. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.
ACS Chemical Biology 06/2007; 2(5):337-46. · 6.45 Impact Factor
-
05/2007;
-
[show abstract]
[hide abstract]
ABSTRACT: Thiolation (T) domains are protein way stations in natural product assembly lines. In the enterobactin synthetase, the T domain on EntF is recognized in cis by its catalytic partners: the EntF condensation (C), adenylation (A), and thioesterase (TE) domains. To assess surface features of the EntF T domain recognized by C, A, and TE, regions of the EntF T domain were submitted to shotgun alanine scanning and Ent production selection, which revealed residues that could not be substituted by Ala. EntF mutants bearing Ala in such positions were assayed in vitro for Ent production with EntEB, and for A-T, C-T, and T-TE communications. We concluded that G1027A and M1030A are specifically defective in acyl transfer from T to TE. These residues define an interaction surface between these two in cis domains in an NRPS module.
Chemistry & Biology 09/2006; 13(8):869-79. · 5.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an alpha-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.
Proceedings of the National Academy of Sciences 12/2005; 102(44):15815-20. · 9.68 Impact Factor
