Part:BBa_K5306000

nasF Periplasmic Binding Protein

This basic part encodes the nasF periplasmic binding protein, which is responsible for binding nitrate (NO₃^-) and facilitating its transport into the cell as part of the NasFED transporter complex.

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.

Usage and Biology

The transporter protein is a crucial part in the ANRA pathway. It’s an ATP-binding casette (ABC) transporter enzyme. Thus, to be able to transport the nitrate ions through the membrane, this group of transporters require energy from ATP hydroloysis. This enzyme is composed of three subunits: a periplasmic protein that binds NO₃^-, a transmembrane protein, and a cytoplasmic ATPase attached to the membrane (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). Its high-affinity binding of NO₃^- enables efficient uptake even at low external concentrations.

nasF is the periplasmic binding protein responsible for the attachment to the outer surface of the cell’s inner membrane through a lipid anchor. The transporter enzyme is required for the uptake of NO3- from the extracellular space, and the nasF protein has the function to bind to it with high affinity (Wu & Stewart, 1998, Lin et al., 1994).

The nasFED nitrate transporter complex (Wu & Stewart, 1998)

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

Considerations

To ensure a proper functionality of the transporter enzyme, the sequence encoding nasF should be introduced with the other 2 subunits nasE(BBa_5306001) and nas D (BBa_5306002) as these genes have been found by Wu & Stewart (1998) to work as an operon. 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 (This part)
• nasE (BBa_5306001)
• nasD (BBa_5306002)
• nasC (BBa_5306003)
• nasB (BBa_5306004)
• nasA (BBa_5306005)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 409
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 409
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 409
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 409
  Illegal AgeI site found at 502
  
• 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/