Difference between revisions of "Part:BBa K5306002"

 
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<h1>nasD Dimeric ATP-Binding Protein </h1>
  
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__NOTOC__
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This basic part encodes the nasD, the cytoplasmic protein subunit subunit of the NasFED transporter complex in the ANRA pathway.
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<partinfo>BBa_K5306002 short</partinfo>
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Nitrate assimilation in bacteria occurs through 3 main steps: (1) Nitrate uptake from the extracellular space by a trasporter, (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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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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nasD constitutes the ATP-binding cytoplasmic component subunit of the transporter complex.
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<h2> Usage and Biology </h2>
  
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<!-- Add more about the biology of this part here
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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. 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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===Usage and Biology===
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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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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5306002 SequenceAndFeatures</partinfo>
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<html><img src = "https://static.igem.wiki/teams/5306/nasfed.jpg"></html>
  
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The nasFED nitrate transporter complex (Wu & Stewart, 1998)
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To ensure a proper functionality of the transporter enzyme, the sequence encoding nasF should be introduced with the other 2 subunits nasE[https://parts.igem.org/Part:BBa_K5306001 (BBa_5306001)] and nas D [https://parts.igem.org/Part:BBa_K5306002 (BBa_5306002)] 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 (BBa_K5306006) or access each one individually from this list:
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<ul>
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  <li>nasF [https://parts.igem.org/Part:BBa_K5306000 (BBa_5306000)]</li>
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  <li>nasE [https://parts.igem.org/Part:BBa_K5306001 (BBa_5306001)] </li>
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<li>nasD (This Part) </li>
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<li>nasC [https://parts.igem.org/Part:BBa_K5306003 (BBa_5306003)] </li>
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<li>nasB [https://parts.igem.org/Part:BBa_K5306004 (BBa_5306004)] </li>
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<li>nasA [https://parts.igem.org/Part:BBa_K5306005 (BBa_5306005)] </li>
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</ul>
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<span class='h3bb'>
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==Sequence and Features==
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</span>
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<partinfo>BBa_K5306003 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K5306002 parameters</partinfo>
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<partinfo>BBa_K5306003 parameters</partinfo>
 
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<h3> References </h3>
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<ol>
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  <li>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</li>
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  <li>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</li>
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  <li>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</li>
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<li>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</li>
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<li>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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</li>
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</ol>

Revision as of 23:45, 27 September 2024

nasD Dimeric ATP-Binding Protein

This basic part encodes the nasD, the cytoplasmic protein subunit subunit of the NasFED transporter complex in the ANRA pathway.

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 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.

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

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 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 (BBa_K5306006) or access each one individually from this list:


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 743
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 743
    Illegal BamHI site found at 1005
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 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/