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Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79.

Acta crystallographica. Section D, Biological crystallography (2004-05-26)
Ekta G Ahuja, Dimitri V Mavrodi, Linda S Thomashow, Wulf Blankenfeldt
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 angstroms 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 P2(1)2(1)2(1), with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 angstroms. The asymmetric unit contains one dimer of PhzA.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
MES hydrate, ≥99.5% (titration)
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MES, low moisture content, ≥99% (titration)
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MES hydrate, BioPerformance Certified, suitable for cell culture, ≥99.5%
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MES sodium salt, BioReagent, ≥99% (titration), crystalline
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MES solution, BioUltra, for molecular biology, 0.5 M in H2O
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MES sodium salt, BioPerformance Certified, suitable for cell culture
SAFC
MES sodium salt
Sigma-Aldrich
MES hydrate, BioUltra, ≥99.5% (T)