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|Title:||An examination of structural properties and prosthetic group turnover of Escherichia coli acyl carrier protein|
|Author(s):||Keating, David H.|
|Doctoral Committee Chair(s):||Cronan, John E.|
|Department / Program:||Biology, Molecular
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Acyl carrier protein (ACP) is a small, highly conserved protein which has an indispensable role in fatty acid biosynthesis, the carrying of fatty acid intermediates. These intermediates are bound in thioester linkage to a sulfhydryl moiety present on the prosthetic group, 4$\sp\prime$-phosphopantetheine, which is added post-translationally to the protein using CoA as a donor.
Overexpression of E. coli ACP results in a decrease in growth rate and subsequent decline in viability. I show that this toxicity results from accumulation of unmodified ACP (apo-ACP), due to inefficient post-translational modification. The accumulated apo-ACP results in a decreased ability to synthesize phospholipids through inhibition of the activity of glycerol 3-phosphate acyltransferase, the first committed step of phospholipid synthesis.
The precursor-product nature of CoA and ACP predicts that depletion of CoA (the ACP prosthetic group donor) should result in the appearance of the unmodified apo form of ACP. However, previous studies were unable to detect such a pool of post-translationally unmodified (apo-ACP) in CoA depleted cells. This result led to the hypothesis that the synthesis of ACP was coupled to the intracellular concentration of CoA, thus preventing synthesis of an inactive (and toxic) form of ACP. The studies presented here demonstrate however, that there is no decrease in ACP transcription under conditions of CoA limitation. The coupling observed previously was an artifactual result of decreased availability of TCA-derived amino acids under CoA-limited conditions.
An altered form of ACP having an unusual mobility on conformationally-sensitive PAGE had previously been observed in strains that carry lesions in fabF, which encodes an isozyme responsible for the condensation step of fatty acid biosynthesis. Reexamination of this altered ACP found it to be the result of a replacement of isoleucine for valine-43. Studies of the mutant protein have shown that its altered mobility is due to a more compact solution structure, suggesting it is a member of the rare class of mutations which result in increased protein stability.
|Rights Information:||Copyright 1996 Keating, David H.|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9625148|