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Comparative study on dihydrofolate reductases from Shewanella species living in deep-sea and ambient atmospheric-pressure environments

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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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Authors and Affiliations

  1. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan

    Chiho Murakami, Eiji Ohmae, Shin-ichi Tate & Kunihiko Gekko

  2. Department of Biotechnology and Chemistry, School of Engineering, Kinki University, Higashi-Hiroshima, 739-2116, Japan

    Kaoru Nakasone

  3. Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, 237-0061, Japan

    Chiaki Kato

Authors
  1. Chiho Murakami
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  2. Eiji Ohmae
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  3. Shin-ichi Tate
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  4. Kunihiko Gekko
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  5. Kaoru Nakasone
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  6. Chiaki Kato
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Correspondence to Eiji Ohmae.

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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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