Exploring and Controlling Phenazine Biosynthesis with Chemical Biology
Final Report Abstract
The phenazines are redox-active bacterial secondary metabolites with functions as virulence factors and respiratory pigments. The most prominent example is the blue phenazine derivative pyocyanin produced by the opportunistic pathogen Pseudomonas aeruginosa. Like the majority of over 150 natural phenazine derivatives known to date, pyocyanin is derived from one of the precursors phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). PCA and PDC are biosynthesized in a conserved pathway that isomerizes chorismate to a reactive aminoketone, of which two then condense to the tricyclic phenazine scaffold before oxidations furnish PCA and PDC in their reduced dihydro forms. The principal steps of phenazine biosynthesis have been unraveled by us and others, using structural and biochemical methods. This laid the basis for the questions addressed in this proposal, which aimed at unveiling mechanistic details of key steps of the pathway by combining chemical biology (supervised by Rolf Breinbauer at Graz Technical University, Austria) with structural biology and biochemistry (supervised by Wulf Blankenfeldt at the Helmholtz Centre for Infection Research and previously at the University of Bayreuth, Germany). In addition to mechanistic studies, we planned to investigate and improve the inhibitory efficacy of tool compounds developed to analyze the Phz-enzymes, since phenazine biosynthesis may be attractive for pharmaceutical intervention. PhzE catalyzes the committing step of the pathway by converting chorismic acid to 2-amino- 2-desoxyisochorismic acid (ADIC). Interestingly, the active center of PhzE is virtually identical to anthranilate synthases (AS) but, unlike these enzymes, does not catalyze pyruvate elimination. Inert ADIC analogues have been synthesized to study differences between AS and PhzE, but due to the fact that the Blankenfeldt group relocated twice during the funding period these compounds await testing. A unique reaction of phenazine biosynthesis is the conversion of 2,3-dihydro-3-hydroxyanthranilic acid (DHHA) to 6-amino-5-oxo-2-cyclohexene-1-carboxylic acid (AOCHC) by PhzF. This isomerization consists of a [1,5]-hydrogen shift followed by keto/enol-tautomerization. Because the first reaction shows several characteristics of an unprecedented enzyme-catalyzed sigmatropic rearrangement, we have investigated PhzF with specifically deuterated synthetic substrate and by computational methods. While we measured a large primary kinetic isotope effect that hints at the importance of proton abstraction in the process, the computations clearly revealed that the [1,5]-hydrogen shift follows an acid/base rather than a sigmatropic mechanism. PhzB condenses two AOCHC molecules to the tricyclic phenazine scaffold. Prior to this proposal, we had obtained product analogues that bound PhzB with 61 nM affinity. Here, we have synthesized 89 new analogues to improve binding further and explore these compounds as inhibitors of PhzB, which may be an attractive drug target not only because it would deprive pathogens of virulence factors but may also lead to self-poisoning due to the high reactivity of the accumulating AOCHC. None of the new compounds bound PhzB more tightly than the best inhibitor available at the start of the project; however, these molecules allow detailed structure/activity relationship analysis. In addition, crystal structures of complexes reveal high tolerance of PhzB towards chemical substitutions in these compounds. In vitro analysis showed that several compounds are indeed good inhibitors, yet they led to significantly reduced pyocyanin production in P. aeruginosa cell culture only when applied at very high concentrations. This hints at inefficient uptake or highly effective efflux, which will be addressed in follow up studies planned in collaboration with experts in drug delivery. The phz-operon of pseudomonads contains a gene duplication of phzB, termed phzA. This protein is inactive in the reaction catalyzed by PhzB but leads to the formation of PhzAB- heterodimers that may be more active than PhzBB homodimers when the substrate concentration is low. Activity assays developed in the course of the funding period seem to corroborate this hypothesis, but questions regarding the relevance of PhzAB remain. However, RT-qPCR analysis and thermal denaturation studies suggest that heterodimers may be the prevalent form of these enzymes in vivo.
Publications
- (2011). Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE. J Biol Chem 286:18213-21
Li QA, Mavrodi DV, Thomashow LS, Roessle M, Blankenfeldt W
- (2013) The Biosynthesis of Phenazines. In: S. Chincholkar, L. Thomashow (eds.). Microbial Phenazines: Biosynthesis, Agriculture and Health. Springer:1-17
Blankenfeldt W
- (2013). Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp. Environ Microbiol 15:675-86
Mavrodi DV, Parejko JA, Mavrodi OV, Kwak YS, Weller DM, Blankenfeldt W, Thomashow LS
(See online at https://doi.org/10.1111/j.1462-2920.2012.02846.x) - (2013). Trapped intermediates in crystals of the FMN-dependent oxidase PhzG provide insight into the final steps of phenazine biosynthesis. Acta Cryst D69: 1403-13
Xu N, Ahuja EG, Janning P, Mavrodi DV, Thomashow LS, Blankenfeldt W
(See online at https://doi.org/10.1107/S0907444913008354) - (2014) Die Biosynthese der Phenazine, Nachrichten aus der Chemie 62:975-80
Diederich C, Leypold M, Breinbauer R, Blankenfeldt W
(See online at https://doi.org/10.1002/nadc.201490357) - (2014) The structural biology of phenazine biosynthesis. Curr Opin Struct Biol 29:26-33
Blankenfeldt W, Parsons JF
(See online at https://doi.org/10.1016/j.sbi.2014.08.013)
