Expression, and molecular and enzymatic characterization of Cu-containing nitrite reductase from a marine ammonia-oxidizing gammaproteobacterium, Nitrosococcus oceani.
Ammonia-oxidizing bacteria (AOB) remove intracellular nitrite to prevent its toxicity by a nitrifier denitrification pathway involving two denitrifying enzymes, nitrite reductase and nitric oxide reductase. Here, a Cu-containing nitrite reductase from Nitrosococcus oceani strain NS58, a gammaproteobacterial marine AOB, was expressed in Escherichia coli and purified to homogeneity. Sequence homology analysis indicated that the nitrite reductase from N. oceani was phylogenetically closer to its counterparts from denitrifying bacteria than that of the betaproteobacterium Nitrosomonas europaea. The recombinant enzyme was a homotrimer of a 32 kDa subunit molecule. The enzyme was green in the oxidized state with absorption peaks at 455 nm and 575 nm. EPR spectroscopy indicated the presence of type 2 Cu. Molecular activities and the affinity constant for the nitrite were determined to be 1.6×10(3) s(-1) and 52 μM, respectively.
Department of Biological Science, Graduate School of Science, Shizuoka University, 836 Oh-ya, Suruga-ku, Shizuoka 422–8529, Japan.
Keitaro
K
Kondo
Katsuhiko
K
Yoshimatsu
Taketomo
T
Fujiwara
D000641:Ammonia / Q000378:metabolism*; D001483:Base Sequence; D058440:Denitrification; D004926:Escherichia coli / Q000235:genetics; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043370:Nitrosomonas europaea / Q000201:enzymology; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D000641:Ammonia; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing