Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein.
Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).
Silvestrini
M C
MC
Dipartimento di Scienze Biochimihe e Centro di Biologia Molecolare del C.N.R., Università di Roma La Sapienza, Italy.
Cutruzzolà
F
F
D'Alessandro
R
R
Brunori
M
M
Fochesato
N
N
Zennaro
E
E
D001400:Azurin / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016958:Pseudomonas putida / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D001400:Azurin; D009565:Nitrate Reductases; D050901:Nitrate Reductase