Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• The nir operon in Escherichia coli encodes the assimilatory nitrite reductase enzyme (NirB) and its small subunit (NirD).
• Understanding the enzymatic properties and interactions of NirB and NirD is crucial for elucidating nitrogen metabolism pathways.

Purpose of the Study:

• To clone and characterize the recombinant NirB enzyme from Escherichia coli for the first time.
• To investigate the role of the small subunit NirD in modulating NirB activity.
• To determine the optimal conditions and kinetic parameters for NirB enzyme activity.

Main Methods:

• Gene cloning of NirB and NirD into the pET28a vector.
• Recombinant enzyme expression and purification.
• Enzyme activity assays using methyl viologen and sodium dithionite as electron donors.
• Docking modeling to predict interactions between NirB, NirD, and substrates/cofactors.
• Determination of optimal temperature, pH, and kinetic parameters (Km, Vmax).

Main Results:

• NirB exhibits high nitrite reductase activity, which is further enhanced by 10% with the inclusion of NirD at a 1:2 NirB:NirD ratio.
• Enzyme aggregation in the absence of β-mercaptoethanol leads to tetrameric to monomeric conversion, with the monomeric form showing highest activity.
• Optimal enzyme activity was observed at 37°C and pH 7.0.
• Kinetic parameters were determined: Km = 9833 μM and Vmax = 416.67 μmol NO2- reduced min-1 mg-1.
• Enzyme activity was inhibited by nitrate and formate but showed increased stability and 10% higher activity in the presence of 25 mM Cd2+.

Conclusions:

• The study provides the first characterization of the recombinant NirB enzyme from Escherichia coli.
• NirD acts as a positive modulator of NirB activity, enhancing nitrite reduction.
• The monomeric form of NirB is the most active, and its stability is influenced by factors like β-mercaptoethanol and cadmium ions.
• These findings contribute to a deeper understanding of the structure-function relationships and regulatory mechanisms of bacterial nitrite reductase.