Coding

Part:BBa_K5306005

Designed by: Iulia Beres   Group: iGEM24_MSP-Maastricht   (2024-09-26)

nasA Molybdoenzyme

This basic part encodes the nasA catalytic subunit of nitrite reductase from the ANRA pathway(Lin et al., 1994).

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 & Stewart, 1997).

Usage and Biology

Assimilatory nitrate reductase catalyzes the reduction of nitrate to nitrite once it has been trasported into the intracellular space. It is composed of two subunits: one α-subunit encoded in the nasA and one β-subunit, in addition to one molybdenum-molybdopterin (Mo-MPT) prosthetic group (Coelho & Romão, 2015). nasA encodes for the protein subunit that catalyzes the reduction of nitrate to nitrite.The Mo-MPT cofactor is the active site of the enzyme and is made by one central molybdenum metal atom, chelated by sulphur in the two molybdopterin cofactors and one oxo ligand (Coelho & Romão, 2015). The molybdenum central atom passes between the +4 and the +6 oxidation states, undergoing a reductive elimination to activate itself and an oxidative addition to execute the NO3-, reduction. The NO3-, substrate is converted into NO2-, by the cycle displayed in the figure. Nitrate reductase uses this mechanism to avoid the formation of harmful byproducts, such as nitric oxide (NO), that can affect the organism.

Image 1
Reduction reaction of nitrate (NO3-) to nitrite (NO2-) by Nitrate Reductase enzyme (Made in ChemDraw)

The coding sequence for nasA was derived from the organism Klebsiella Oxytoca M5al by Wu & Stewart(1998) and has undergone codon optimization for Vibrio natriegens.

Considerations

To ensure a proper functionality of the nitrate reductase enzyme, the sequence encoding nasA should be introduced with the other subunit nasC(BBa_5306003). 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:


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 230
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1011
    Illegal PstI site found at 230
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2423
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 230
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 230
    Illegal AgeI site found at 1797
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. Coelho, C. and Romão, M.J. (2015), Structural and mechanistic insights on nitrate reductases. Protein Science, 24: 1901-1911. https://doi.org/10.1002/pro.2801
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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/
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