The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes ofPseudomonas aeruginosaCatalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.
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 inPseudomonas aeruginosaEnzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate thatP. aeruginosacontains 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 reductionin 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 genelpdGAlthough 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.
From the Divisions of Biology and Biological Engineering and.
Nathaniel R
NR
Glasser
From the Divisions of Biology and Biological Engineering and.
Benjamin X
BX
Wang
From the Divisions of Biology and Biological Engineering and.
Julie A
JA
Hoy
From the Divisions of Biology and Biological Engineering and dkn@caltech.edu.|Geology and Planetary Sciences, California Institute of Technology, Pasadena, California 91125.
Dianne K
DK
Newman
D001426:Bacterial Proteins / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D009243:NAD; D010084:Oxidation-Reduction; D010619:Phenazines / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011710:Pyocyanine / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
D001426:Bacterial Proteins; D010619:Phenazines; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; C037165:1-phenazinecarboxylic acid; D011710:Pyocyanine; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase
Pseudomonas aeruginosa (P. aeruginosa); X-ray crystallography; dihydrolipoamide dehydrogenase; enzyme kinetics; metabolism; phenazine; pyruvate dehydrogenase complex (PDC)