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Intein‐mediated creation of a cyclic acyl carrier protein
Author(s) -
Volkmann Gerrit,
Murphy Peter W.,
Rowland Elden E.,
Liu XiangQin,
Byers David M.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.850.8
Subject(s) - acyl carrier protein , acyl group , intein , chemistry , biochemistry , acylation , enzyme , acyltransferase , stereochemistry , circular dichroism , biosynthesis , gene , organic chemistry , rna , alkyl , rna splicing , catalysis
Acyl carrier protein (ACP) is an essential protein in bacterial metabolism, where it acts as the major donor for fatty acyl groups in the synthesis of lipids and other specialized products. As such, ACP is an attractive anti‐microbial drug target. ACP normally encloses attached fatty acyl groups in a hydrophobic pocket within its three mainα‐helices, but the exact mechanism of interaction and acyl transfer from ACP to partner enzymes is poorly understood. ACP interacts with most proteins through its acidic helix II, and acyl transfer must involve partial or full unfolding of ACP in order to present the acyl chain to the partner enzyme. To examine the requirement of ACP unfolding for acyl transfer, we created a cyclic version of Vibrio harveyi ACP using split‐intein technology. Mass spectrometry, circular dichroism and fluorescence studies suggest that cyclic ACP (cACP) is trapped in a folded conformation. In vitro characterization indicates that cACP can be modified by holo‐ACP synthetase yielding an active ACP. Furthermore, in vivo complementation assays suggest that cACP is able to replace the linear wild‐type ACP and support growth. Our results thus suggest that cACP is able to interact with and transfer its acyl chain to its many partner enzymes within the bacterial cell despite constraining of its N‐ and C‐termini. (Supported by CIHR, NSERC, and an IWK Studentship).