Abstract
The major O2-insensitive nitroreductase (NfsA) of Escherichia coli shares low sequence homology but similar biochemical and structural features with NfsB, the E. coli minor O2-insensitive nitroreductase. A structural comparison revealed Phe42 was present in the active site of NfsA but not NfsB. F42Y, F42N and F42A were generated and had decreased activity toward nitrofurazone by 52, 96, and 99 %, respectively. The kinetic parameters for other nitroaromatic substrates were also determined. Compared to wild type, the mutants did not have significantly altered K ms, but had dramatically decreased k cat and k cat/K m values. Far-UV CD spectral analysis of the mutants suggested that there were no significant conformational changes however F42A and F42N had changes from 208 to 222 nm, which was attributed to loss of helix content. These findings revealed that Phe42 is important for maintaining NfsA activity and structure.
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The authors acknowledge the financial support provided by the National Key Project for Basic Research (2010CB126102), the National High Technology Research and Development Program of China (2011AA10A204), Science and Technology Project of Liaoning Province (2012225113), The Fundamental Research Funds for the Central Universities (DUT13JB08) and Dalian Municipal Science and Technology Program (2013E13SF114).
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Yang, J., Zhan, J., Bai, J. et al. Residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA. Biotechnol Lett 35, 1693–1700 (2013). https://doi.org/10.1007/s10529-013-1262-y
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DOI: https://doi.org/10.1007/s10529-013-1262-y