| RRC ID |
3276
|
| 著者 |
Lai JR, Fischbach MA, Liu DR, Walsh CT.
|
| タイトル |
Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection.
|
| ジャーナル |
J Am Chem Soc
|
| Abstract |
Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4'-phosphopantetheine prosthetic group), the specific protein-protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB-ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB-ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB-ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB-ArCP in the enterobactin synthetase and that the majority of EntB-ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.
|
| 巻・号 |
128(34)
|
| ページ |
11002-3
|
| 公開日 |
2006-8-30
|
| DOI |
10.1021/ja063238h
|
| PMID |
16925399
|
| MeSH |
Escherichia coli Proteins / chemistry*
Escherichia coli Proteins / genetics
Models, Molecular
Mutagenesis*
Protein Binding
Selection, Genetic*
Surface Properties
|
| IF |
14.612
|
| 引用数 |
40
|
|
WOS 分野
|
CHEMISTRY, MULTIDISCIPLINARY
|
| リソース情報 |
| 原核生物(大腸菌) |
|
