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Thomas Smith's Laboratory |
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The nrt operon
The nrt gene products are responsible for the high affinity, ATP-dependent, transport of nitrate into the cytoplasm. The organization is similar to the metal transporters from 6803 in that it has a solute binding protein (NrtA), a pore protein (NrtB), and an ATPase motor (NrtD). The major difference between the nrt and, for example, the znu systems is the NrtC protein. NrtC is an unusual fusion protein composed of the solute binding domain of NrtA and the ATPase domain of NrtD. How it functions is unclear, but it has been shown to be involved with regulation of nitrate import. The only other protein showing signficant homology to NrtA is the analogous protein from the bicarbonate transport system CmpA. One goal here was to understand the atomic selectivity of the protein for nitrate over bicarbonate.
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Atomic structure of NrtA
We determined the structure of NrtA to a resolution of 1.5Å. The protein is similar to the metal binding proteins such as ZnuA but with a number of significant differences. Like ZnuA, the protein has a general 'C clamp' shape. In this image, you are looking into the 'mouth' of the protein at the bound nitrate molecule. Unlike ZnuA, there are extra helices to the right and left of the mouth that likely negatively affected sequence alignments. Also different from ZnuA is that NrtA does not have a rigid alpha-helix running up the back of the clamp and one would expect it to be more flexible than ZnuA.
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Nitrate binding environment
Shown here is the electron density of the bound nitrate (right) and the atomic details of the nitrate binding environment (left). From sequence alignments, the lysine 269 is changed to an acid in the bicarbonate binding protein, CmpA - and could be a major determinant in ligand specificity. Unexpectly, this acid is not found in the regulatory protein, CmpC. Instead CmpC looks more like NrtA and the regulatory protein for nitrate import, NrtC. From this we suggest that nitrate may be co-regulating nitrate and bicarbonate import. |
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Possible regulation of nitrate/bicarbonate transport
As mentioned above, the solute binding domains of the regulatory proteins of both nitrate and bicarbonate transport proteins, NrtC and CmpC, both seem to bind nitrate over bicarbonate. It is possible that both transporters are regulated by nitrate. As shown below, a lot of energy goes into the reduction of nitrate into ammonium. If there is a buildup of nitrate (or ammonium) then import is shut down to conserve both ATP and reduction potential. Interestingly it seems possible that nitrate might concomitently shut down bicarbonate transport to maintain the proper carbon/nitrogen ratio for the cell. |
Our relevant publications:
- Koropatkin, N. M., Pakrasi, H. B., Smith, T. J. (2006) Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity. PNAS 103:9820-9825.
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