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The pyruvate and α-ketoglutarate dehydrogenase complexes of Pseudomonas aeruginosa catalyze pyocyanin and phenazine-1-carboxylic acid reduction via the subunit dihydrolipoamide dehydrogenase

Glasser, Nathaniel R. and Wang, Benjamin X. and Hoy, Julie A. and Newman, Dianne K. (2017) The pyruvate and α-ketoglutarate dehydrogenase complexes of Pseudomonas aeruginosa catalyze pyocyanin and phenazine-1-carboxylic acid reduction via the subunit dihydrolipoamide dehydrogenase. Journal of Biological Chemistry, 292 (13). 5593-5607. ISSN 0021-9258. PMCID PMC5392700. http://resolver.caltech.edu/CaltechAUTHORS:20170213-091715272

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Abstract

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M116.772848DOIArticle
http://www.jbc.org/content/292/13/5593PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392700/PubMed CentralArticle
ORCID:
AuthorORCID
Newman, Dianne K.0000-0003-1647-1918
Additional Information:© 2017 The American Society for Biochemistry and Molecular Biology. Received December 16, 2016. Accepted February 7, 2017. First Published on February 7, 2017. We thank Dr. Doug Rees and the Caltech Molecular Observatory for providing essential guidance, training, and materials for X-ray crystallography; the Caltech Proteome Exploration Laboratory for its services; and the SSRL staff. Members of the Newman laboratory (past and present) provided constructive feedback on the manuscript. This work was supported in part by Howard Hughes Medical Institute, National Institutes of Health Grant 5R01HL117328-03, National Science Foundation Grant 1144469, and the Amgen Scholars Program. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author Contributions: N. R. G. and D. K. N. designed the experiments and wrote the manuscript. N. R.G. collected the data and refined the protein structures. B. X. W. created the protein expression vectors, assisted with protein purification, and performed preliminary enzyme kinetics. B. X. W. and J. A. H. performed the initial crystallography screens and optimized crystal growth conditions. J. A. H. discovered the high resolution crystal form of LpdG and provided crystallography training. N. R. G. further optimized crystallization and cryoprotection. D. K. N. coordinated the project. All authors reviewed the manuscript.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH5R01HL117328-03
NSF Graduate Research FellowshipDGE-1144469
Amgen Scholars ProgramUNSPECIFIED
Subject Keywords:enzyme kinetics, metabolism, Pseudomonas aeruginosa, pyruvate dehydrogenase complex (PDC), X-ray crystallography, dihydrolipoamide dehydrogenase, phenazine
PubMed Central ID:PMC5392700
Record Number:CaltechAUTHORS:20170213-091715272
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20170213-091715272
Official Citation:Nathaniel R. Glasser, Benjamin X. Wang, Julie A. Hoy, and Dianne K. Newman The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase J. Biol. Chem. 2017 292: 5593-. doi:10.1074/jbc.M116.772848
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:74227
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:13 Feb 2017 20:13
Last Modified:02 Mar 2018 21:48

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