NotesFAQContact Us
Search Tips
ERIC Number: ED526611
Record Type: Non-Journal
Publication Date: 2009
Pages: 126
Abstractor: As Provided
Reference Count: 0
ISBN: ISBN-978-1-1095-8038-9
Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase
Thomas, Jacob
ProQuest LLC, Ph.D. Dissertation, University of Illinois at Urbana-Champaign
Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes this prosthetic group was discovered over 40 years ago in "Escherichia coli" and was soon followed by the observation that the ACP prosthetic group turns over independently of the protein "in vivo." However, the gene encoding this enzyme, its structural properties and physiological function were unknown. I have identified the gene encoding ACP phosphodiesterase in "Escherichia coli" and named it "acpH." I have constructed a deletion mutant lacking AcpH, demonstrating that it is nonessential for growth but required for ACP turnover both "in vitro" and "in vivo." I have also purified the C-terminally hexahistidine-tagged AcpH enzyme and shown that it is active on holo-ACP as well as acyl-ACP thioesters with acyl chain lengths of 6-16 carbons. It is also able to use some non-native acyl carrier proteins as substrates. I have also used bioinformatic methods to investigate the structural properties of AcpH in collaboration with Professor Daniel J. Rigden of the University of Liverpool. Homology modeling suggested that AcpH is related to the hydrolase domain of the Rel/Spo guanosine tetraphosphate synthetase/hydrolase of Streptococcus equisimilis. Residues predicted to be important for Mn[superscript 2+] binding were mutagenically altered and found to be important for enzyme activity. The activity of some mutants could also be partially restored by the addition of an excess of Mn[superscript 2+]. These results allowed us to conclude that AcpH is a member of the HD family of phosphohydrolases despite lacking the characteristic histidine of the conserved motif. The activity of AcpH was also examined in relation to the toxicity of "N"-pentylpantothenamide, an analog of the CoA precursor pantothenic acid. This analog is known to be a substrate for three CoA synthetic enzymes, resulting in a CoA analog with a metabolically inactive prosthetic group (ethyldethia-CoA) which is then transferred to ACP. It was thought that ethyldethia-ACP carrying this inactive prosthetic group was a poor substrate for AcpH, resulting in the selective turnover of holo-ACP. I have shown that this is not the case: ethyldethia-ACP is a substrate for AcpH and overexpression of this enzyme results in increased resistance to "N"-pentylpantothenamide. I have also shown that CoA synthesis is inhibited upon treatment with the analog and this is likely to be a major cause of its growth-inhibitory effect. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page:]
ProQuest LLC. 789 East Eisenhower Parkway, P.O. Box 1346, Ann Arbor, MI 48106. Tel: 800-521-0600; Web site:
Publication Type: Dissertations/Theses - Doctoral Dissertations
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A