| RRC ID |
3275
|
| 著者 |
Lai JR, Fischbach MA, Liu DR, Walsh CT.
|
| タイトル |
A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection.
|
| ジャーナル |
Proc Natl Acad Sci U S A
|
| Abstract |
Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis.
|
| 巻・号 |
103(14)
|
| ページ |
5314-9
|
| 公開日 |
2006-4-4
|
| DOI |
10.1073/pnas.0601038103
|
| PII |
0601038103
|
| PMID |
16567620
|
| PMC |
PMC1459352
|
| MeSH |
Amino Acid Sequence
Base Sequence
DNA Primers
Models, Molecular
Molecular Sequence Data
Mutagenesis
Peptides / chemistry*
Point Mutation
Ribosomal Proteins / biosynthesis*
Ribosomal Proteins / chemistry
Selection, Genetic*
Sequence Homology, Amino Acid
|
| IF |
9.412
|
| 引用数 |
51
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
JW0587 |
