Cold Adaptation of Triosephosphate Isomerase.

The main problem for enzymes from psychrophilic species, which need to work near the freezing point of liquid water, is the exponential decay of reaction rates as the temperature is decreased. Cold-adapted enzymes have solved this problem by shifting the activation enthalpy-entropy balance for the catalyzed reaction compared to those of their mesophilic orthologs. To understand the structural basis of this universal feature, it is necessary to examine pairs of such orthologous enzymes, with known three-dimensional structures, at the microscopic level. Here, we use molecular dynamics free energy calculations in combination with the empirical valence bond method to evaluate the temperature dependence of the activation free energy for differently adapted triosephosphate isomerases. The results show that the enzyme from the psychrophilic bacterium Vibrio marinus indeed displays the characteristic shift in enthalpy-entropy balance, compared to that of the yeast ortholog. The origin of this effect is found to be located in a few surface-exposed protein loops that show differential mobilities in the two enzymes. Key mutations render these loops more mobile in the cold-adapted triosephosphate isomerase, which explains both the reduced activation enthalpy contribution from the protein surface and the lower thermostability.

Extreme biophysics: Enzymes under pressure.

A critical question about piezophilic (pressure-loving) microbes is how their constituent molecules maintain function under high pressure. Here, factors are examined that may lead to the increased activity under pressure in dihydrofolate reductase from the piezophilic Moritella profunda compared to the homologous enzyme from the mesophilic Escherichia coli. Molecular dynamics simulations are performed at various temperatures and pressures to examine how pressure affects the flexibility of the enzymes from these two microbes, since both stability and flexibility are necessary for enzyme activity. The results suggest that collective motions on the 10-ns timescale are responsible for the flexibility necessary for "corresponding states" activity at the growth conditions of the parent organism. In addition, the results suggest that while the lower stability of many enzymes from deep-sea microbes may be an adaptation for greater flexibility at low temperatures, high pressure may enhance their adaptation to low temperatures. © 2017 Wiley Periodicals, Inc.

The role of interfacial lipids in stabilizing membrane protein oligomers.

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Structure of a complete four-domain chitinase from Moritella marina, a marine psychrophilic bacterium.

X-ray crystallography reveals chitinase from the psychrophilic bacterium Moritella marina to be an elongated molecule which in addition to the catalytic ß/α-barrel domain contains two Ig-like domains and a chitin-binding domain, all linked in a chain. A ligand-binding study using NAG oligomers showed the enzyme to be active in the crystal lattice and resulted in complexes of the protein with oxazolinium ion (the reaction intermediate) and with NAG2, a reaction product. The characteristic motif DXDXE, containing three acidic amino-acid residues, which is a signature of type 18 chitinases, is conserved in the enzyme. Further analysis of the unliganded enzyme with the two protein-ligand complexes and a comparison with other known chitinases elucidated the roles of other conserved residues near the active site. Several features have been identified that are probably important for the reaction mechanism, substrate binding and the efficiency of the enzyme at low temperatures. The chitin-binding domain and the tryptophan patch on the catalytic domain provide general affinity for chitin, in addition to the affinity of the binding site; the two Ig-like domains give the protein a long reach over the chitin surface, and the flexible region between the chitin-binding domain and the adjacent Ig-like domain suggests an ability of the enzyme to probe the surface of the substrate, while the open shallow substrate-binding groove allows easy access to the active site.

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.

Structural studies of the lipopolysaccharide of Moritella viscosa strain M2-226.

The structure of the O-specific side chain of the lipopolysaccharide from the Gram-negative psychrophilic bacterium Moritella viscosa strain M2-226, responsible for the winter ulcer in Atlantic salmon, has been determined. Monosaccharide analysis and (1)H and (13)C NMR spectroscopy were employed to elucidate the structure. It was concluded that the polysaccharide is composed of a trisaccharide repeating unit with the following structure: →3)-ß-D-GlcpNAc-(1→4)-[α-D-GlcpA-(1→3)]-α-L-Fucp-(1→ .

Reduced susceptibility of Moritella profunda dihydrofolate reductase to trimethoprim is not due to glutamate 28.

The E28D variant of dihydrofolate reductase from Moritella profunda was generated and found to have the same K (i) (within error) for the competitive inhibitor trimethoprim as the wild type enzyme. Contrary to a previous claim in the literature, Glu 28 is therefore not the cause of the reduced affinity for trimethoprim relative to dihydrofolate reductase from Escherichia coli.

Moritella profunda sp. nov. and Moritella abyssi sp. nov., two psychropiezophilic organisms isolated from deep Atlantic sediments.

Strains 2674T (=LMG 21259T =JCM 11435T) and 2693T (=LMG 21258T =JCM 11436T) were isolated from Atlantic sediments at a temperature of 2 degrees C and a depth of 2815 m off the West African coast. Polyphasic evidence indicates that the two strains belong to the genus Moritella and represent distinct species, for which the names Moritella profunda sp. nov. (for strain 2674T) and Moritella abyssi sp. nov. (for strain 2693T) are proposed. The moderate piezophily of the two organisms is intermediate between that of the type species, Moritella marina, which is not piezophilic, and Moritella yayanosii, an obligate piezophile. Both are strict psychrophiles with slightly different cardinal temperatures: at 0.1 MPa, maximal growth rates are observed at 2 degrees C (M. profunda) and 4 degrees C (M. abyssi) with maximum temperatures of 12 degrees C (M. profunda) or 14 degrees C (M. abyssi). The optimal pressure is lower than that at the site of isolation, and raising the temperature to 10 degrees C makes the organisms more piezophilic.