Difference between revisions of "Part:BBa K5306002"
 
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<h1>nasD Dimeric ATP-Binding Protein </h1>
 
<h1>nasD Dimeric ATP-Binding Protein </h1>
  
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This basic part encodes the nasD, the cytoplasmic protein subunit subunit of the NasFED transporter complex in the ANRA pathway (Lin & Stewart, 1997).  
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This basic part encodes the nasD, the cytoplasmic protein subunit of the NasFED transporter complex in the ANRA pathway (Lin & Stewart, 1997).  
  
 
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.  
 
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.  
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<h2> Usage and Biology </h2>
 
<h2> Usage and Biology </h2>
  
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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 (Lin & Stewart, 1997). This enzyme is composed of of three subunits: a periplasmic protein that binds NO<sub>3</sub><sup>-</sup>, a transmembrane protein, and a cytoplasmic ATPase attached to the membrane (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). Its high-affinity binding of NO<sub>3</sub><sup>-</sup> enables efficient uptake even at low external concentrations.
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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 (Lin & Stewart, 1997). This enzyme is composed of three subunits: a periplasmic protein that binds NO<sub>3</sub><sup>-</sup>, a transmembrane protein, and a cytoplasmic ATPase attached to the membrane (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). Its high-affinity binding of NO<sub>3</sub><sup>-</sup> enables efficient uptake even at low external concentrations.
  
 
nasD constitutes the ATP-binding cytoplasmic component subunit of the transporter complex. This protein is responsible for the hydrolysis of ATP to ADP that will provide the energy necessary for the uptake of the nitrate ions.  
 
nasD constitutes the ATP-binding cytoplasmic component subunit of the transporter complex. This protein is responsible for the hydrolysis of ATP to ADP that will provide the energy necessary for the uptake of the nitrate ions.  
  
 
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<img height="200px" width="300px" src="https://static.igem.wiki/teams/5306/nasfed.jpg" alt="Image 1">
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<img height="200px" width="300px" src="https://static.igem.wiki/teams/5306/bba-k5306000-3nasfed.jpg" alt="Image 1">
 
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</img>
 
</html>
 
</html>
  
 
The nasFED nitrate transporter complex (Wu & Stewart, 1998)
 
The nasFED nitrate transporter complex (Wu & Stewart, 1998)
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The coding sequence of nasD is derived from the organism <i> Klebsiella Oxytoca M5al </i> by Wu and Stewart (1998) and has undergone codon optimization for <i> Vibrio natriegens </i>.
  
 
<h2> Considerations </h2>
 
<h2> Considerations </h2>
  
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To ensure a proper functionality of the transporter enzyme, the sequence encoding nasD should be introduced with the other 2 subunits nasF[https://parts.igem.org/Part:BBa_K5306000 (BBa_5306000)] and nas E [https://parts.igem.org/Part:BBa_K5306001 (BBa_5306001)] as these genes has 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 [https://parts.igem.org/Part:BBa_K5306006 (BBa_5306006)] or access each one individually from this list:
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To ensure a proper functionality of the transporter enzyme, the sequence encoding nasD should be introduced with the other 2 subunits nasF[https://parts.igem.org/Part:BBa_K5306000 (BBa_5306000)] and nas E [https://parts.igem.org/Part:BBa_K5306001 (BBa_5306001)] 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 [https://parts.igem.org/Part:BBa_K5306006 (BBa_5306006)] or access each one individually from this list:
  
 
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<ul>

Latest revision as of 11:20, 1 October 2024

nasD Dimeric ATP-Binding Protein

This basic part encodes the nasD, the cytoplasmic protein subunit of the NasFED transporter complex in the ANRA pathway (Lin & Stewart, 1997).

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 (Lin & Stewart, 1997). 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.

nasD constitutes the ATP-binding cytoplasmic component subunit of the transporter complex. This protein is responsible for the hydrolysis of ATP to ADP that will provide the energy necessary for the uptake of the nitrate ions.

Image 1

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

The coding sequence of nasD 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 nasD should be introduced with the other 2 subunits nasF(BBa_5306000) and nas E (BBa_5306001) 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:


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 58
    Illegal XbaI site found at 483
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 58
    Illegal NheI site found at 708
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 58
    Illegal XhoI site found at 367
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 58
    Illegal XbaI site found at 483
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 58
    Illegal XbaI site found at 483
  • 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/