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 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 screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.
authors: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607-7173, USA. khevener@uic.edu
authors: Kirk E
authors: KE
authors: Hevener
authors: Shahila
authors: S
authors: Mehboob
authors: Teuta
authors: T
authors: Boci
authors: Kent
authors: K
authors: Truong
authors: Bernard D
authors: BD
authors: Santarsiero
authors: Michael E
authors: ME
authors: Johnson
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D005486:Flavin Mononucleotide; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)