Similar structural stabilities of 3-isopropylmalate dehydrogenases from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 and its atmospheric congener S. oneidensis strain MR-1.

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ±â€¯0.1 MPa and a volume change of -225 ±â€¯24 mL mol-1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and -30 mL mol-1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

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

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.

Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli Dihydrofolate Reductase and Its Mutants: Contributions of Phenylalanine and Tyrosine Side Chains and Exciton Coupling of Two Tryptophan Side Chains.

Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy has recently been used for secondary structure analysis of proteins; however, the contribution of aromatic side chains to protein VUV CD spectra is unresolved. In this report, VUV CD spectra of 10 Escherichia coli dihydrofolate reductase (DHFR) mutants, in which each phenylalanine or tyrosine residue was mutated to leucine, were measured down to 175 nm at 25 °C and pH 8.0 to elucidate the contributions of these aromatic side chains to the high-energy transitions of peptide bonds. The VUV CD spectra of these mutants were different from the spectrum of the wild-type protein, indicating that the contribution of the phenylalanine and tyrosine side chains of DHFR extends to the VUV region. Furthermore, the VUV CD spectrum and the folate- or NADP(+)-induced spectral change of F103L mutant DHFR indicated a modification and regeneration of exciton coupling between the Trp47 and Trp74 side chains, respectively, suggesting that exciton coupling may also contribute to the CD spectrum of DHFR in the VUV region. These results should be useful for theoretically characterizing the contribution of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy.

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.

Effects of salt on the structure, stability, and function of a halophilic dihydrofolate reductase from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1.

The effects of salt on the structure, stability, and enzymatic function of a novel dihydrofolate reductase (HjDHFR P1) from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1 living in a Japanese saltern, were studied using ultraviolet absorption, circular dichroism (CD), and fluorescence spectroscopy. HjDHFR P1 had a partial structure at pH 8.0 in the absence of NaCl, and the addition of NaCl (0-500 mM concentration) induced significant structural formation to HjDHFR P1. The addition of NADPH, which is a coenzyme for its catalytic reaction, and lowering the pH from 8 to 6 also induced the same CD change, indicating the formation of the NADPH-binding site in HjDHFR P1. The NaCl dependence of thermal and urea-induced unfolding measurements suggested that protein stability increased depending on NaCl concentration regardless of structural formation, and HjDHFR P1 achieved the same stability as Escherichia coli DHFR at 750 mM NaCl. Halophilic characteristics were also observed for enzymatic function, although its structure had already formed under the conditions that enzymatic activity was measured at due to the presence of NADPH. These results suggest that the halophilic mechanism on structural stability and function was caused by factors other than structural formation, which are suggested to be the contributions of preferential interactions between the protein and salt ions and the specific binding of salt ions.

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.

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.

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, 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.

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants.

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Delta52 in an alphaC-betaC loop. Double deletion mutants showed that the loop alphaC-betaC has nonadditive couplings with the betaF-betaG and betaG-betaH loops regarding stability. Single deletion to the loops alphaC-betaC or betaC-betaD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.

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.

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.

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.

Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions.

Methionine-42, distal to the active site of Escherichia coli dihydrofolate reductase, was substituted by site-directed mutagenesis with 14 amino acids (Ala, Cys, Glu, Gln, Gly, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr, and Val) to elucidate its role in the stability and function of this enzyme. Far-ultraviolet circular dichroism spectra of these mutants showed a distinctive negative peak at around 230 nm beside 220 nm, depending on the hydrophobicity of the amino acids introduced. The fluorescence intensity also increased in an order similar to that of the amino acids. These spectroscopic data suggest that the mutations do not affect the secondary structure, but strongly perturb the exciton coupling between Trp47 and Trp74. The free energy of urea unfolding, deltaG(o)u, increased with increases in the side-chain hydrophobicity in the range 2.96-6.40 kcal x mol(-1), which includes the value for the wild-type enzyme (6.08 kcal x mol(-1)). The steady-state kinetic parameters, Km and kcat, also increased with increases in the side-chain hydrophobicity, with the M42W mutant showing the largest increases in Km (35-fold) and kcat (4.3-fold) compared with the wild-type enzyme. These results demonstrate that site 42 distal to the active site plays an important role in the stability and function of this enzyme, and that the main effect of the mutations is to modify of hydrophobic interactions with the residues surrounding this position.

Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase.

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

Mass spectrometry of hydrogen/deuterium exchange of Escherichia coli dihydrofolate reductase: effects of loop mutations.

To address the effects of single amino acid substitutions on the structural fluctuation of Escherichia coli dihydrofolate reductase (DHFR), hydrogen/deuterium exchange kinetics were investigated at 15 degrees C with wild-type and mutant DHFRs at Gly67 (six mutants) and Gly121 (eight mutants) located in two flexible loops, by means of electrospray ionization mass spectrometry. These mutations induced significant changes in the first-order rate constant of proton exchange, k(ex) (0.10-0.27 min(-1)), the number of fast-exchangeable protons, Delta M(o) (164-222 Da), and the number of protons protected from exchange, Delta M(infinity) (15-56 Da), relative to the corresponding values for the wild-type enzyme (k(ex) = 0.18 min(-1), Delta M(o) = 164 Da, and Delta M(infinity) = 50.5 Da). These kinetic parameters were strongly correlated with the volume of introduced amino acids, but partly correlated with adiabatic compressibility (volume fluctuation), stability, and enzymatic activity. These results indicate that the local structure change due to a single amino acid substitution in loop regions is dramatically magnified to affect the structural fluctuation of the whole DHFR molecule, resulting in complicated changes in its stability and function.