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Journal of Bacteriology, June 2009, p. 3804-3810, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00046-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

{alpha}-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant {alpha}-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens{triangledown}

Ryan J. Mailloux, Ranji Singh, Guy Brewer, Christopher Auger, Joseph Lemire, and Vasu D. Appanna*

Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada

Received 14 January 2009/ Accepted 8 April 2009

{alpha}-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH4) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. 13C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada. Phone: (705) 675-1151, ext. 2112. Fax: (705) 675-4844. E-mail: vappanna{at}laurentian.ca

{triangledown} Published ahead of print on 17 April 2009.

Journal of Bacteriology, June 2009, p. 3804-3810, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00046-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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