title: Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein.
abstract: 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).
authors.affiliation: Dipartimento di Scienze Biochimihe e Centro di Biologia Molecolare del C.N.R., Università di Roma La Sapienza, Italy.
authors.forename: M C
authors.initials: MC
authors.lastname: Silvestrini
authors.forename: F
authors.initials: F
authors.lastname: Cutruzzolà
authors.forename: R
authors.initials: R
authors.lastname: D'Alessandro
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: N
authors.initials: N
authors.lastname: Fochesato
authors.forename: E
authors.initials: E
authors.lastname: Zennaro
mesh_terms: 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
chemical_list: D011994:Recombinant Proteins; D001400:Azurin; D009565:Nitrate Reductases; D050901:Nitrate Reductase