The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Abstract

Edited by Joseph JezPhenazines 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 hemecontaining 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 ␣-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or ␣-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.

The Colorful World of Extracellular Electron Shuttles

“…Recent work from our laboratory shows that cytoplasmic (flavo)proteins can catalyze phenazine reduction in P. aeruginosa (46). Specifically, the enzyme dihydrolipoamide dehydrogenase enables phenazines to substitute for NAD + in the pyruvate and α-ketoglutarate dehydrogenase complexes.…”

“…Specifically, the enzyme dihydrolipoamide dehydrogenase enables phenazines to substitute for NAD + in the pyruvate and α-ketoglutarate dehydrogenase complexes. This activity may allow phenazines to promote ATP synthesis during glucose oxidation by increasing the flux through pyruvate dehydrogenase and acetate kinase (46) (Figure 4 b ). Similar findings have been observed in fermenting organisms, where the presence of humic substances or analogs increases the ratio of oxidized-to-reduced products (7), presumably increasing the ATP yield of fermentation.…”

“…( b ) A model of phenazine reduction in Pseudomonas aeruginosa that allows for energy conservation by electron shuttling. By substituting for NAD + in the pyruvate dehydrogenase complex (PDH), phenazines enable the synthesis of acetyl-CoA, which can drive ATP synthesis through the enzymes phosphate transacetylase and acetate kinase (45, 46). Efflux of reduced phenazines by MexGHI-OpmD (100) may be coupled to proton translocation.…”

“…In this review, we focus our discussion primarily on colorful, endogenous EESs, drawing upon our experience studying phenazines produced by Pseudomonas aeruginosa (20, 21, 30–33, 45, 46, 54, 55, 58, 93–95, 115, 119–121) (Figure 2 c ). We use phenazines for illustrative purposes only to pique the reader’s curiosity about what other molecules may have similar physiological functions.…”

ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway inPseudomonas aeruginosa

“…TPP-dependent enzymes including transketolase, pyruvate dehydrogenase, 2oxoglutarate dehydrogenase, and 1-deoxy-dxylulose-5-phosphate synthase, catalyze essential cellular reactions in bacteria, from central metabolism to biosynthesis of amino acids, cofactors, and lipids (21). Recently, TPPdependent pyruvate and α-ketoglutarate dehydrogenase complexes in P. aeruginosa were found to intracellularly reduce phenazin derivatives such as pyocyanin (a pseudomonal toxin), thus contributing to iron acquisition and redox homeostasis of the bacteria in addition to their designated roles in central metabolism (41). This evidence indicates that TPP deficiency can have a substantial effect on the metabolic network of bacteria, thus making TPP metabolism a very attractive target for the development of new antibiotics.…”