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Phza/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis Ekta G

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Phza/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis Ekta G Subscriber access provided by DigiTop | USDA's Digital Desktop Library Article PhzA/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis Ekta G. Ahuja, Petra Janning, Matthias Mentel, Almut Graebsch, Rolf Breinbauer, Wolf Hiller, Burkhard Costisella, Linda S. Thomashow, Dmitri V. Mavrodi, and Wulf Blankenfeldt J. Am. Chem. Soc., 2008, 130 (50), 17053-17061 • DOI: 10.1021/ja806325k • Publication Date (Web): 17 November 2008 Downloaded from http://pubs.acs.org on January 15, 2009 More About This Article Additional resources and features associated with this article are available within the HTML version: • Supporting Information • Access to high resolution figures • Links to articles and content related to this article • Copyright permission to reproduce figures and/or text from this article Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published on Web 11/17/2008 PhzA/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis Ekta G. Ahuja,† Petra Janning,† Matthias Mentel,‡,§ Almut Graebsch,‡ Rolf Breinbauer,†,‡,§,| Wolf Hiller,‡ Burkhard Costisella,‡ Linda S. Thomashow,⊥,# Dmitri V. Mavrodi,⊥ and Wulf Blankenfeldt*,† Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany, Technical UniVersity of Dortmund, Faculty of Chemistry, Otto-Hahn-Strasse 6, 44221 Dortmund, Germany, UniVersity of Leipzig, Institute of Organic Chemistry, Johannisallee 29, 04103 Leipzig, Germany, Graz UniVersity of Technology, Institute of Organic Chemistry, Stremayrgasse 16, 8010 Graz, Austria, Washington State UniVersity, Pullman, Washington 99164-6430, and USDA, Agricultural Research SerVice, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 Received August 10, 2008; E-mail: [email protected] Abstract: Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the ∆5-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine- 1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives. 1. Introduction model of lung infections.2,3 Since the lungs of nearly all victims of cystic fibrosis are chronically colonized by P. aeruginosa The production of phenazine pigments is among the most and this infection contributes significantly to the low life readily observable phenomena in bacteria, due to their intense expectancy of these patients,4 phenazine production may present color. This pigmentation triggered the early discovery of the an attractive target for pharmaceutical intervention. blue phenazine derivative pyocyanin (1-hydroxy-5N-meth- Phenazines play multiple roles in the bacteria that generate ylphenazinium betaine), which was isolated in the 1850s from them, and only recently has an understanding of these roles wound dressings of soldiers infected with Pseudomonas aerugi- begun to emerge.5 Phenazine-producing strains residing on roots nosa.1 Phenazines are redox-active and can reduce molecular can protect cultivated plants against susceptible bacterial and oxygen, leading to the generation of toxic reactive oxygen fungal pathogens.6,7 In soil, phenazines can participate in iron + species and explaining their broad-spectrum antibiotic activity. acquisition by reducing environmental Fe3 to the more soluble 2+ They also act as virulence factors in human infectious disease. Fe , and they also function in primary energy metabolism by For example, pyocyanin induces apoptosis in neutrophils, and strains of Pseudomonas aeruginosa defective in pyocyanin (2) Allen, L.; Dockrell, D. H.; Pattery, T.; Lee, D. G.; Cornelis, P.; Hellewell, P. G.; Whyte, M. K. J. Immunol. 2005, 174, 3643–3649. synthesis are more susceptible to immune responses in a mouse (3) Lau, G. W.; Hassett, D. J.; Ran, H.; Kong, F. Trends Mol. Med. 2004, 10, 599–606. (4) Heijerman, H. J. Cyst. Fibros. 2005, 4 Suppl 2, 3–5. † Max-Planck-Institute of Molecular Physiology. (5) Price-Whelan, A.; Dietrich, L. E.; Newman, D. K. Nat. Chem. Biol. ‡ Technical University of Dortmund. 2006, 2, 71–78. § University of Leipzig. (6) Giddens, S. R.; Feng, Y.; Mahanty, H. K. Mol. Microbiol. 2002, 45, | Graz University of Technology. 769–783. ⊥ Washington State University. (7) Cook, R. J.; Thomashow, L. S.; Weller, D. M.; Fujimoto, D.; Mazzola, # USDA. M.; Bangera, G.; Kim, D. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, (1) Fordos, J. Recl. TraV. Soc. d’Emulation Sci. Pharm. 1859, 3,30. 4197–4201. 10.1021/ja806325k CCC: $40.75 2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 17053–17061 9 17053 ARTICLES Ahuja et al. Scheme 1. Biosynthesis of Phenazine-1-carboxylic acid (7)a a Steps upstream of PhzA/B catalysis are shown in green, and intermediates discussed here are shown in red. Orange indicates byproducts. Reactions that lead to the formation of PhzA/B’s product are boxed. mediating the reoxidation of NADH and utilization of pyruvate last intermediate that can be isolated. In the next step, PhzF under oxygen-limiting conditions.8,9 In P. aeruginosa, pyocyanin isomerizes 316,17 to 6-amino-5-oxocyclohex-2-ene-1-carboxylic is a signaling molecule that controls gene expression by directly acid (4) by initiating a [1,5]-prototropic shift.16 This ketone can activating the iron-containing transcription factor SoxR.10 Col- undergo spontaneous dimerization by symmetrical head-to-tail lectively, these observations reveal some of the ways in which double condensation in Vitro, yielding a tricyclic phenazine phenazines are exploited by their producers to interact with the precursor 5/5′/5a that must be further oxidized to arrive at the environment, showing that they are more than simple secondary aromatic phenazine moiety. In Pseudomonas, the steps past metabolites. ketone condensation include oxidative decarboxylation to 6, and Dozens of naturally occurring phenazine derivatives have core phenazine biosynthesis ends at 7, whereas other bacteria, been isolated, and existing knowledge indicates that all of them e.g. Pantoea agglomerans, generate 9 as their core phenazine.6 originate from either phenazine-1-carboxylic acid (7) or phena- Intriguingly, in Vitro studies have revealed that none of the steps zine-1,6-dicarboxylic acid (9) (Scheme 1).11-13 These “core” following ketone formation require enzyme catalysis, and PhzF phenazines are synthesized from two molecules of chorismic alone can trigger conversion of 3 to 7, albeit with low yield.16,17 acid (1) in a pathway catalyzed by enzymes encoded in the This raises questions about the function of the remaining conserved “phz operon”. A compilation of available sequence conserved enzymes PhzA/B and PhzG. While it is relatively data indicates that five Phz-enzymes are indispensable for production of 7 and/or 9 (Scheme 1 and Supporting Information (14) McDonald, M.; Mavrodi, D. V.; Thomashow, L. S.; Floss, H. G. J. Am. Chem. Soc. 2001, 123, 9459–9460. Figure S1). Of these, PhzE converts 1 to 2-amino-4-desoxy- (15) Parsons, J. F.; Calabrese, K.; Eisenstein, E.; Ladner, J. E. Biochemistry isochorismate (2).14 Second, isochorismatase PhzD cleaves 2 2003, 42, 5684–5693. to generate trans-2,3-dihydro-3-hydroxyanthranilate (3),15 the (16) Blankenfeldt, W.; Kuzin, A. P.; Skarina, T.; Korniyenko, Y.; Tong, L.; Bayer, P.; Janning, P.; Thomashow, L. S.; Mavrodi, D. V. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16431–16436. (8) Hernandez, M. E.; Kappler, A.; Newman, D. K. Appl. EnViron. (17) Parsons, J. F.; Song, F.; Parsons, L.; Calabrese, K.; Eisenstein, E.; Microbiol. 2004, 70, 921–928. Ladner, J. E. Biochemistry 2004, 43, 12427–12435. (9) Price-Whelan, A.; Dietrich, L. E.; Newman, D. K. J. Bacteriol. 2007, (18) Parsons, J. F.; Calabrese, K.; Eisenstein, E.; Ladner, J. E. Acta 189, 6372–6381. Crystallogr., D: Biol. Crystallogr. 2004, 60, 2110–2113. (10) Dietrich, L. E.; Price-Whelan, A.; Petersen, A.; Whiteley, M.; Newman, (19) Mavrodi, D. V.; Ksenzenko, V. N.; Bonsall, R. F.; Cook, R. J.; D. K. Mol. Microbiol. 2006, 61, 1308–1321. Boronin, A. M.; Thomashow, L. S. J. Bacteriol. 1998, 180, 2541– (11) Turner, J. M.; Messenger, A. J. AdV. Microb. Physiol. 1986, 27, 211– 2548.
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