Abstract
A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme’s eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.
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Abbreviations
- CD:
-
Circular dichroism
- EPR:
-
Electron paramagnetic resonance
- IPTG:
-
Isopropyl-ß-d-thiogalactopyranoside
- LB:
-
Luria-Bertani
- MALDI-TOF:
-
Matrix-assisted laser desorption ionization-time of flight
- NBS:
-
N-bromosuccinimide
- SDS-PAGE:
-
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
- WT:
-
Wild-type
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Acknowledgments
The authors would like to thank Prof. Steven Rothstein (University of Guelph) for a generous gift of a spinach nitrite reductase clone, Prof. Toshiharu Hase (University of Osaka) for a generous gift of a clone of the E. coli cysG gene and for many helpful discussions. This research was supported by a grant (to DBK) from the U.S. Department of Energy (DE-FG02-99ER20346) and a grant (to JFA) from the U.S. Department of Agriculture (2003-02149).
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Tripathy, J.N., Hirasawa, M., Kim, SK. et al. The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach. Photosynth Res 94, 1–12 (2007). https://doi.org/10.1007/s11120-007-9198-5
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DOI: https://doi.org/10.1007/s11120-007-9198-5