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Title: Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

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 mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US 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 amore » 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{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.« less

Authors:
; ; ; ; ;  [1]
  1. (UIC)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
UNIVERSITYNIH
OSTI Identifier:
1048969
Resource Type:
Journal Article
Journal Name:
Protein Expres. Purif.
Additional Journal Information:
Journal Volume: 85; Journal Issue: (1) ; 09, 2012
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; CARBOXYLIC ACIDS; CLONING; CRYSTALLIZATION; DESIGN; ENZYMES; FLUORESCENCE; OXIDOREDUCTASES; PATHOGENS; PIPELINES; PROTEINS; PURIFICATION; RESOLUTION; SYNTHESIS; TARGETS; THERAPY

Citation Formats

Hevener, Kirk E., Mehboob, Shahila, Boci, Teuta, Truong, Kent, Santarsiero, Bernard D., and Johnson, Michael E. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis. United States: N. p., 2012. Web. doi:10.1016/j.pep.2012.07.003.
Hevener, Kirk E., Mehboob, Shahila, Boci, Teuta, Truong, Kent, Santarsiero, Bernard D., & Johnson, Michael E. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis. United States. doi:10.1016/j.pep.2012.07.003.
Hevener, Kirk E., Mehboob, Shahila, Boci, Teuta, Truong, Kent, Santarsiero, Bernard D., and Johnson, Michael E. Thu . "Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis". United States. doi:10.1016/j.pep.2012.07.003.
@article{osti_1048969,
title = {Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis},
author = {Hevener, Kirk E. and Mehboob, Shahila and Boci, Teuta and Truong, Kent and Santarsiero, Bernard D. and Johnson, Michael E.},
abstractNote = {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 mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US 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{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.},
doi = {10.1016/j.pep.2012.07.003},
journal = {Protein Expres. Purif.},
number = (1) ; 09, 2012,
volume = 85,
place = {United States},
year = {2012},
month = {10}
}