title: Comparative study on dihydrofolate reductases from Shewanella species living in deep-sea and ambient atmospheric-pressure environments.
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.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Chiho
authors.initials: C
authors.lastname: Murakami
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohmae
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tate
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Gekko
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nakasone
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D001272:Atmosphere; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011312:Pressure; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology / Q000235:genetics*; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013816:Thermodynamics; D014871:Water Microbiology
chemical_list: D013762:Tetrahydrofolate Dehydrogenase