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Expression, Purification and Characterization of Enoyl‐ACP Reductase II, FabK, from Porphyromonas gingivalis

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Title: Expression, Purification and Characterization of Enoyl‐ACP Reductase II, FabK, from Porphyromonas gingivalis
Author(s): Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.
Subject(s): Porphyromonas gingivalis FabK enoyl-ACP reductase II purification characterization crystallization
Abstract: The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS‐II, are distinct from their mammalian counterparts, FAS‐I, in terms of both structure and enzymatic mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. One such enzyme, enoyl‐acyl carrier protein (ACP) reductase II, FabK, is a key, rate‐limiting enzyme in the FAS‐II pathway. The bacterial organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the U.S. population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS‐II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping‐Pong Bi‐Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for high‐throughput, 384‐well format. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x‐rays to high resolution.
Issue Date: 2012-09
Publisher: Elsevier
Citation Info: Hevener KE, Mehboob S, Boci T, Truong K, Santarsiero BD, Johnson ME. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis. Protein Expression and Purification. 2012 Sep;85(1):100-8. doi: 10.1016/j.pep.2012.07.003
Type: Article
Description: NOTICE: This is the author’s version of a work that was accepted for publication in Protein Expression and Purification. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Protein Expression and Purification, Vol 85, Issue 1, (2012) DOI: 10.1016/j.pep.2012.07.003
URI: http://hdl.handle.net/10027/10708
ISSN: 1096-0279
Sponsor: Support for this research was provided by the UIC College of Dentistry MOST program, NIH T32DE018381
Date Available in INDIGO: 2013-11-26
 

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