The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa.
The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 A, alpha = gamma = 90, beta = 110.5 degrees. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 A. Seleno-L-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 A, alpha = gamma = 90, beta = 110.5 degrees and the diffraction limit is 2.7 A.
Gohain
Neelakshi
N
Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Thomashow
Linda S
LS
Mavrodi
Dmitri V
DV
Blankenfeldt
Wulf
W
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011710:Pyocyanine / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D011710:Pyocyanine; D006899:Mixed Function Oxygenases; C439053:PhzS protein, Pseudomonas aeruginosa