Journal
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume 82, Issue 10, Pages 2691-2702Publisher
WILEY-BLACKWELL
DOI: 10.1002/prot.24635
Keywords
structural biology; siderophores; acyl-carrier proteins; enzyme structure; enterobactin; natural product biosynthesis; secondary metabolism
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Funding
- National Institutes of Health [GM-068440]
- Hauptman-Woodward Institute (Stafford Fellowship)
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Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. (C) 2014 Wiley Periodicals, Inc.
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