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Title: OVEREXPRESSION, PURIFICATION AND CRYSTALLIZATION OF PHZA, THE FIRST ENZYME OF THE PHENAZINE BIOSYNTHESIS PATHWAY OF PSEUDOMONAS FLUORESCENS 2-79.

Author
item AHUJA, E - MAX-PLANK-INSTITUTE
item MAVRODI, D - WASHINGTON STATE UNIV
item Thomashow, Linda
item BLANKENFELDT, W - MAX-PLANK-INSTITUTE

Submitted to: Acta Crystallographica Section B: Structural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2004
Publication Date: 7/20/2004
Citation: Ahuja, E.G., Mavrodi, D.V., Thomashow, L.S., Blankenfeldt, W. 2004. Overexpression, purification and crystallization of phza, the first enzyme of the phenazine biosynthesis pathway of pseudomonas fluorescens 2-79. Acta Crystallographica B. D60: 1129-1131.

Interpretive Summary: Phenazines are broad-spectrum antibiotic metabolites produced by bacteria such as Pseudomonas and Streptomyces. Phenazines have a broad role in the ecology of the biocontrol agents Pseudomonas fluorescens and P. aureofaciens, and are a factor in the virulence of the human pathogen P. aeruginosa. In P. fluorescens and P. aureofaciens, phenazines are responsible for biocontrol activity against soilborne pathogens that attack plant roots and they contribute to the competitiveness of these bacteria in the rhizosphere of wheat. Although phenazines have been known since before 1900, the mechanism of synthesis remains unclear. We previously cloned seven phenazine biosynthetic genes from P. fluorescens 2-79 and showed that they are conserved among all phenazine-producing fluorescent Pseudomonas species. Here, we have overexpressed one of these genes, phzA, and crystallized the protein as a first step towards understanding its role in the synthesis of phenazine compounds. Understanding the mechanism of synthesis of these compounds is important not only because of their role in plant protection, but also because despite their potential pharmaceutical value there are presently no commercially attractive ways to synthesize them.

Technical Abstract: Phenazines are broad-spectrum antibiotic metabolites produced by organisms such as Pseudomonas and Streptomyces. Phenazines have been shown to enhance microbial competitiveness and the pathogenic potential of the organisms that synthesize them. PhzA (163 residues, approximate molecular weight 18.7 kDa) is the product of the first of seven genes of the operon responsible for phenazine biosynthesis in P. fluorescens 2-79. This enzyme is thought to catalyse one of the final steps in the formation of phenazine-1-carboxylic acid, the end product of phenazine biosynthesis in P. fluorescens 2-79. Here, the purification and crystallization of recombinant PhzA are reported. Crystals diffracting to 2.1 Å were obtained using 1.6 M magnesium sulfate and 2-morpholinoethanesulfonic acid monohydrate (MES) buffer pH 5.2-5.6. Crystals of both native and seleno-L-methionine-labelled protein belong to the orthorhombic space group P212121, with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 Å. The asymmetric unit contains one dimer of PhzA.