Eiji Ohmae

Acetonitrile-protein interactions: amino acid solubility and preferential solvation

The solubility of amino acids and the preferential solvent interaction of hen-egg lysozyme in acetonitrile (AN)-water mixtures (<60 w/v% AN) were investigated by means of densimetric and refractometric methods at 25 degreesC. The free energy of transfer from water to aqueous AN was negative for most nonpolar side-chains of amino acids and positive for the peptide group, the extent being comparable to those for methanol and ethanol systems. Addition of AN to an aqueous solvent was thus suggested to weaken the hydrophobic interaction and to enhance the peptide-peptide hydrogen bond therein leading to the denaturation of proteins. A parallel examination by circular dichroism confirmed that the conformation of lysozyme (pH 3) remains native in aqueous AN up to 40% but changes to the helix-rich form at higher AN concentrations. At all solvent compositions up to 50% AN (pH 3), however, lysozyme was preferentially hydrated probably due to a local salting-out of the AN molecules from the charges on the protein surface, indicating the increase of the chemical potential of the protein. These results are discussed in relation to the role of AN as an eluting organic solvent in reverse-phase chromatography.

Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.

Cloning and characterization of dihydrofolate reductases from deep-sea bacteria

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

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

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, Km and kcat; although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in kcat/Km) 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.

Thermodynamic and functional characteristics of deep-sea enzymes revealed by pressure effects.

Hydrostatic pressure analysis is an ideal approach for studying protein dynamics and hydration. The development of full ocean depth submersibles and high-pressure biological techniques allows us to investigate enzymes from deep-sea organisms at the molecular level. The aim of this review was to overview the thermodynamic and functional characteristics of deep-sea enzymes as revealed by pressure axis analysis after giving a brief introduction to the thermodynamic principles underlying the effects of pressure on the structural stability and function of enzymes.

Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli

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.

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.

Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution

Abstract 3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

Pressure dependence of the apparent specific volume of bovine serum albumin: Insight into the difference between isothermal and adiabatic compressibilities

There are some theoretical arguments related to interpreting the adiabatic compressibility (beta(s)) of a protein determined from the sound velocity and the difference between beta(s) and isothermal compressibility (beta(T)). To address these problems experimentally, we constructed a high-pressure oscillating densitometer and used it to measure the apparent specific volume of bovine serum albumin as a function of pressure (0.1-78MPa) and temperature (5-35 degrees C). The beta(T) determined from plots of the apparent specific volume vs. pressure was slightly larger than beta(s) at all temperatures examined, with the difference between the two compressibilities increasing as the temperature was decreased. Only at room temperature did the observed beta(T) agree with those estimated from beta(s) using the heat capacity and the thermal expansibility of the protein, suggesting that there are significant as-yet-unknown mechanisms that affect protein compressibility.

Environmental Adaptation of Dihydrofolate Reductase from Deep-Sea Bacteria

In order to elucidate the molecular adaptation mechanisms of enzymes to the high hydrostatic pressure of the deep sea, we cloned, purified, and characterized more than ten dihydrofolate reductases (DHFRs) from bacteria living in deep-sea and ambient atmospheric pressure environments. The nucleotide and amino acid sequences of these DHFRs indicate the deep-sea bacteria are adapted to their environments after the differentiation of their genus from ancestors inhabiting atmospheric pressure environments. In particular, the backbone structure of the deep-sea DHFR from Moritella profunda (mpDHFR) almost overlapped with the normal homolog from Escherichia coli (ecDHFR). Thus, those of other DHFRs would also overlap on the basis of their sequence similarities. However, the structural stability of both DHFRs was quite different: compared to ecDHFR, mpDHFR was more thermally stable but less stable against urea and pressure unfolding. The smaller volume changes due to unfolding suggest that the native structure of mpDHFR has a smaller cavity and/or enhanced hydration compared to ecDHFR. High hydrostatic pressure reduced the enzymatic activity of many DHFRs, but three deep-sea DHFRs and the D27E mutant of ecDHFR exhibited pressure-dependent activation. The inverted activation volumes from positive to negative values indicate the modification of their structural dynamics, conversion of the rate-determining step of the enzymatic reaction, and different contributions of the cavity and hydration to the transition-state structure. Since the cavity and hydration depend on amino acid side chains, DHFRs would adapt to the deep-sea environment by regulating the cavity and hydration by substituting their amino acid side chains without altering their backbone structure. The results of this study clearly indicate that the cavity and hydration play important roles in the adaptation of enzymes to the deep-sea environment.