scholarly journals Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres

1993 ◽  
Vol 296 (3) ◽  
pp. 885-885 ◽  
Keyword(s):  
Nitrite Reductase ◽  
Purification And Characterization ◽  
Dissimilatory Nitrite Reductase

scholarly journals The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (8) ◽  
pp. 2323-2329 ◽  
Author(s):  
Miguel Prudêncio ◽  
Robert R. Eady ◽  
Gary Sawers
Keyword(s):  
Amino Acid ◽  
Nitrite Reductase ◽  
Recombinant Enzyme ◽  
Leader Peptide ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

scholarly journals Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

2001 ◽  
Vol 183 (14) ◽  
pp. 4149-4156 ◽  
Author(s):  
Hirotaka Ichiki ◽  
Yoko Tanaka ◽  
Kiyotaka Mochizuki ◽  
Katsuhiko Yoshimatsu ◽  
Takeshi Sakurai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Nitrite Reductase ◽  
Sodium Dodecyl ◽  
Resonance Spectroscopy ◽  
Halophilic Archaeon ◽  
Haloarcula Marismortui ◽  
Dissimilatory Nitrite Reductase

ABSTRACT Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (gII = 2.232; AII = 4.4 mT) and type 2 Cu centers (gII = 2.304; AII = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO2 − · min−1 · mg−1.

Dissimilatory nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) is trimeric and contains both type 1 and type 2 copper centres.

1993 ◽  
Vol 51 (1-2) ◽  
pp. 359
Author(s):  
Z. Abraham ◽  
E.T. Adman ◽  
T. Brüser ◽  
R.R. Eady ◽  
J.G. Grossmann ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Dissimilatory Nitrite Reductase

scholarly journals Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres

1993 ◽  
Vol 295 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Z H L Abraham ◽  
D J Lowe ◽  
B E Smith
Keyword(s):  
Nitrite Reductase ◽  
Specific Activity ◽  
Visible Region ◽  
Exchange Chromatography ◽  
Published Data ◽  
Absorption Bands ◽  
Sds Page ◽  
Dissimilatory Nitrite Reductase

Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this ‘blue’ nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.

scholarly journals Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients.

1987 ◽  
Vol 84 (23) ◽  
pp. 8628-8632 ◽  
Author(s):  
G. J. Cooper ◽  
A. C. Willis ◽  
A. Clark ◽  
R. C. Turner ◽  
R. B. Sim ◽  
...  
Keyword(s):  
Diabetic Patients ◽  
Purification And Characterization ◽  
Type 2 Diabetic Patients ◽  
Type 2 Diabetic
Sign in / Sign up

Export Citation Format

Share Document