Effect of Arginine at Type 2 Cu Site on Spectroscopic Features and Enzymatic Activity of Copper-containing Nitrite Reductase

2007 ◽  
Vol 36 (1) ◽  
pp. 140-141
Author(s):  
Kazuya Yamaguchi ◽  
Aiko Tsuda ◽  
Masaki Nojiri ◽  
Shinnichiro Suzuki
Keyword(s):  
Enzymatic Activity ◽  
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 Oral DhHP-6 for the Treatment of Type 2 Diabetes Mellitus

2019 ◽  
Vol 20 (6) ◽  
pp. 1517 ◽  
Author(s):  
Kai Wang ◽  
Yu Su ◽  
Yuting Liang ◽  
Yanhui Song ◽  
Liping Wang
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Blood Glucose ◽  
Enzymatic Activity ◽  
Glucose Levels ◽  
Blood Glucose Levels

Type 2 diabetes mellitus (T2DM) is associated with pancreatic β-cell dysfunction which can be induced by oxidative stress. Deuterohemin-βAla-His-Thr-Val-Glu-Lys (DhHP-6) is a microperoxidase mimetic that can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we demonstrated an increased stability of linear peptides upon their covalent attachment to porphyrins. In this study, we assessed the utility of DhHP-6 as an oral anti-diabetic drug in vitro and in vivo. DhHP-6 showed high resistance to proteolytic degradation in vitro and in vivo. The degraded DhHP-6 product in gastrointestinal (GI) fluid retained the enzymatic activity of DhHP-6, but displayed a higher permeability coefficient. DhHP-6 protected against the cell damage induced by H2O2 and promoted insulin secretion in INS-1 cells. In the T2DM model, DhHP-6 reduced blood glucose levels and facilitated the recovery of blood lipid disorders. DhHP-6 also mitigated both insulin resistance and glucose tolerance. Most importantly, DhHP-6 promoted the recovery of damaged pancreas islets. These findings suggest that DhHP-6 in physiological environments has high stability against enzymatic degradation and maintains enzymatic activity. As DhHP-6 lowered the fasting blood glucose levels of T2DM mice, it thus represents a promising candidate for oral administration and clinical therapy.

Characterization of Azide-Binding Type 2 Cu(II) Site of Nitrite Reductase

2001 ◽  
Vol 74 (1) ◽  
pp. 183-184
Author(s):  
Kunishige Kataoka ◽  
Shinnichiro Suzuki
Keyword(s):  
Nitrite Reductase ◽  
Azide Binding ◽  
Binding Type

Catalytic Function and Local Proton Structure at the Type 2 Copper of Nitrite Reductase:  The Correlation of Enzymatic pH Dependence, Conserved Residues, and Proton Hyperfine Structure†

Biochemistry ◽  
2002 ◽  
Vol 41 (23) ◽  
pp. 7464-7474 ◽  
Author(s):  
Yiwei Zhao ◽  
Dmitriy A. Lukoyanov ◽  
Yuriy V. Toropov ◽  
Kenneth Wu ◽  
James P. Shapleigh ◽  
...  
Keyword(s):  
Hyperfine Structure ◽  
Nitrite Reductase ◽  
Ph Dependence ◽  
Conserved Residues ◽  
Proton Structure ◽  
Catalytic Function

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.

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