Abstract
To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74–90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K m and k cat; although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k cat/K m) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.
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Abbreviations
- DHF:
-
Dihydrofolate
- DHFR:
-
Dihydrofolate reductase
- ecDHFR:
-
DHFR from Escherichia coli
- sb21DHFR:
-
DHFR from Shewanella benthica strain DB21MT-2
- sb43992DHFR:
-
DHFR from S. benthica strain ATCC43992
- sb6705DHFR:
-
DHFR from S. benthica strain DB6705
- sfDHFR:
-
DHFR from S. frigidimarina strain ACAM591
- sgDHFR:
-
DHFR from S. gelidimarina strain ACAM456
- soDHFR:
-
DHFR from S. oneidensis strain MR-1
- spDHFR:
-
DHFR from S. putrefaciens strain IAM12079
- svDHFR:
-
DHFR from S. violacea strain DSS12
- THF:
-
Tetrahydrofolate
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Acknowledgments
This work was financially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (no. 16657031 for K.G.).
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Communicated by F. Robb.
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Murakami, C., Ohmae, E., Tate, Si. et al. Comparative study on dihydrofolate reductases from Shewanella species living in deep-sea and ambient atmospheric-pressure environments. Extremophiles 15, 165–175 (2011). https://doi.org/10.1007/s00792-010-0345-0
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DOI: https://doi.org/10.1007/s00792-010-0345-0