title: Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli.
abstract: To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80°C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25°C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.
authors.affiliation: Department of Mathematical and Life Sciences, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan. ohmae@hiroshima-u.ac.jp
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohmae
authors.forename: Chiho
authors.initials: C
authors.lastname: Murakami
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tate
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Gekko
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hata
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Akasaka
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kato
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D006874:Hydrostatic Pressure; D007700:Kinetics; D034221:Moritella / Q000737:chemistry* / Q000201:enzymology; D009792:Oceans and Seas; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D014867:Water / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014867:Water; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase