Part:BBa_K5306004

nasB Nitrite Reductase

This basic part encodes the nasB nitrite reductase enzyme which is responsible for reducing nitrate to ammonium in the ANRA pathway (Lin et al., 1993).

Assimilatory nitrate reduction to ammonium (ANRA) in bacteria occurs through 3 main steps: (1) Nitrate uptake from the extracellular space by a transporter, (2) Conversion of nitrate to nitrite by the nitrate reductase enzyme and (3) Reduction to ammonium of the nitrite by nitrite reductase (Lin et al., 1993, Lin et al., 1994).

Usage and Biology

The nitrite reductase enzyme encoded by nasB, is part of the cytochrome enzymes family and utilizes multiple heme groups to catalyze the conversion of NO₂^- to NH₄⁺ (Lin et al., 1993). These heme groups, which contain a central iron atom that alternates between oxidation states of +2 and +3, are tetracoordinated to a protoporphyrin IX ring. This ring stabilizes the +3 oxidation state during the reduction of NO₂^- through electron delocalization (Einsle et al., 2002). The reduction process begins with the oxidative addition of NO₂^- to the iron center, resulting in the loss of one oxygen atom as water and the formation of a hydroxylamine intermediate, which subsequently transforms into an amine through electron transfers involving NADPH (Einsle et al., 2002). In the final stages, the amine undergoes double protonation, allowing it to be reductively eliminated from the metal center as the final product, NH₄⁺ (Einsle et al., 2002). This enzyme was selected because its reaction mechanism avoids the formation of a hydrazine intermediate, a highly toxic, carcinogenic, and explosive compound that could pose significant risks to the cell.

The reduction reaction of nitrite (NO₂^-) to ammonia (NH₄⁺) (Made in ChemDraw)

The coding sequence for nasB was derived from Klebsiella Oxytoca M5al by Wu & Stewart(1998) and has been codon optimized for our chassis V. natriegens .

Considerations

The ANRA pathway's enzymes have been found by Lin et al. (1994) to function as an operon, thus, a 6x HIS tag was inserted at the 3' end of the coding sequence for nasB to be able to locate the protein and confirm the expression of the three enzymes when introduced together.

We recommend that nasB to be expressed together with the other proteins to ensure proper functionality of the pathway. If you want to introduce the whole nitrate assimilation pathway as our team did, you can find the whole operon sequence that contains all the coding sequences that you need in our composite part’s page (BBa_5306006) or access each one individually from this list:

• nasF ((BBa_5306000)
• nasE (BBa_5306001)
• nasD (BBa_5306002)
• nasC (BBa_5306003)
• nasB (This Part)
• nasA (BBa_5306005)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 471
  Illegal XbaI site found at 1821
  Illegal PstI site found at 713
  Illegal PstI site found at 2222
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 318
  Illegal NheI site found at 822
  Illegal NheI site found at 1851
  Illegal PstI site found at 713
  Illegal PstI site found at 2222
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 745
  Illegal BglII site found at 2160
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 471
  Illegal XbaI site found at 1821
  Illegal PstI site found at 713
  Illegal PstI site found at 2222
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 471
  Illegal XbaI site found at 1821
  Illegal PstI site found at 713
  Illegal PstI site found at 2222
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Lee, H. H., Ostrov, N., Wong, B. G., Gold, M. A., Khalil, A. S., & Church, G. M. (2019). Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. *Nature Microbiology*, *4*(7), 1105–1113. https://doi.org/10.1038/s41564-019-0423-8
2. Lin, J. T., Goldman, B. S., & Stewart, V. (1993). Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al. *Journal of Bacteriology*, *175*(8), 2370–2378. https://doi.org/10.1128/jb.175.8.2370-2378.1993
3. Lin, J. T., Goldman, B. S., & Stewart, V. (1994). The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al. *Journal of Bacteriology*, *176*(9), 2551–2559. https://doi.org/10.1128/jb.176.9.2551-2559.1994
4. Moreno-Vivián, C., & Flores, E. (2007, January 1). *Chapter 17 - Nitrate Assimilation in Bacteria* (H. Bothe, S. J. Ferguson, & W. E. Newton, Eds.). ScienceDirect; Elsevier. https://www.sciencedirect.com/science/article/abs/pii/B9780444528575500187?via%3Dihub
5. Wu, Q., & Stewart, V. (1998). NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in Klebsiella oxytoca (pneumoniae) M5al. *Journal of Bacteriology*, *180*(5), 1311–1322. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107022/
6. Einsle, O., Messerschmidt, A., Huber, R., Peter, & Neese, F. (2002). Mechanism of the Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase. Journal of the American Chemical Society, 124(39), 11737–11745. https://doi.org/10.1021/ja0206487