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Temperature dependence of the flexibility of thermophilic and mesophilic flavoenzymes of the nitroreductase fold.

ABSTRACT: A widely held hypothesis regarding the thermostability of thermophilic proteins states asserts that, at any given temperature, thermophilic proteins are more rigid than their mesophilic counterparts. Many experimental and computational studies have addressed this question with conflicting results. Here, we compare two homologous enzymes, one mesophilic (Escherichia coli FMN-dependent nitroreductase; NTR) and one thermophilic (Thermus thermophilus NADH oxidase; NOX), by multiple molecular dynamics simulations at temperatures from 5 to 100 degrees C. We find that the global rigidity/flexibility of the two proteins, assessed by a variety of metrics, is similar on the time scale of our simulations. However, the thermophilic enzyme retains its native conformation to a much greater degree at high temperature than does the mesophilic enzyme, both globally and within the active site. The simulations identify the helix F-helix G 'arm' as the region with the greatest difference in loss of native contacts between the two proteins with increasing temperature. In particular, a network of electrostatic interactions holds helix F to the body of the protein in the thermophilic protein, and this network is absent in the mesophilic counterpart.

SUBMITTER: Merkley ED

PROVIDER: S-EPMC2851445 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Temperature dependence of the flexibility of thermophilic and mesophilic flavoenzymes of the nitroreductase fold.

Merkley Eric D ED Parson William W WW Daggett Valerie V

Protein engineering, design & selection : PEDS 20100118 5

A widely held hypothesis regarding the thermostability of thermophilic proteins states asserts that, at any given temperature, thermophilic proteins are more rigid than their mesophilic counterparts. Many experimental and computational studies have addressed this question with conflicting results. Here, we compare two homologous enzymes, one mesophilic (Escherichia coli FMN-dependent nitroreductase; NTR) and one thermophilic (Thermus thermophilus NADH oxidase; NOX), by multiple molecular dynamic ...[more]

PMID: 20083491

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Project description:The occurrence of non-isoprenoid alkyl glycerol ether lipids in Bacteria and natural environments is increasingly being reported and the specificity and diagenetic stability of these lipids make them powerful biomarkers for biogeochemical and environmental studies. Yet the environmental controls on the biosynthesis of these peculiar membrane lipids remain poorly documented. Here, the lipid content of two mesophilic (Desulfatibacillum aliphaticivorans and Desulfatibacillum alkenivorans) and one thermophilic (Thermodesulfobacterium commune) sulfate-reducing bacteria-whose membranes are mostly composed of ether lipids-was investigated as a function of growth temperature (20-40°C and 54-84°C, respectively). For all strains, the cellular lipid content was lower at sub- or supra-optimal growth temperature, but the relative proportions of dialkyl glycerols, monoalkyl glycerols and fatty acids remained remarkably stable whatever the growth temperature. Rather than changing the proportions of the different lipid classes, the three strains responded to temperature changes by modifying the average structural composition of the alkyl and acyl chains constitutive of their membrane lipids. Major adaptive mechanisms concerned modifications of the level of branching and of the proportions of the different methyl branched lipids. Specifically, an increase in temperature induced mesophilic strains to produce less dimethyl branched dialkyl glycerols and 10-methyl branched lipids relative to linear structures, and the thermophilic strain to decrease the proportion of anteiso relative to iso methyl branched compounds. These modifications were in agreement with a regulation of the membrane fluidity. In one mesophilic and the thermophilic strains, a modification of the growth temperature further induced changes in the relative proportions of sn-2 vs sn-1 monoalkyl glycerols, suggesting an unprecedented mechanism of homeoviscous adaptation in Bacteria. Strong linear correlations observed between different ratios of alkyl glycerols and temperature allow to hypothesize the use of these specific lipids as indicators of temperature changes in the environment.

Project description:The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

Dataset Information

Temperature dependence of the flexibility of thermophilic and mesophilic flavoenzymes of the nitroreductase fold.

Publications

Temperature dependence of the flexibility of thermophilic and mesophilic flavoenzymes of the nitroreductase fold.

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