Abstract
The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10°C to 70°C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function.
Highlights
A steadily increasing demand by the biotechnology industry exists to discover and make available enzymes with high stability and catalytic efficiency at both elevated and low temperatures
We have investigated the structure–function relationship of four homologous enzymes originating from psychrophilic, psychrotrophic, mesophilic and thermophilic bacteria to study enzyme adaptation to extreme temperatures
Out of all bacterial ester hydrolases (EC 3.1) with an α/β-hydrolase fold available in the PDB, the following enzymes were excluded: (a) enzymes from pathogenic organisms, (b) membrane-bound enzymes, (c) enzymes with low-resolution structures (>3 Å), (d) enzyme structures without primary literature data being available, (e) heteromultimeric enzymes and (f ) enzymes with structures solved by methods other than X-ray crystallography
Summary
A steadily increasing demand by the biotechnology industry exists to discover and make available enzymes with high stability and catalytic efficiency at both elevated and low temperatures (de Miguel Bouzas et al, 2006; Joshi and Satyanarayana, 2013). (thermophiles) or below 20°C ( psychrophiles) are a valuable source of such enzymes (Basu and Sen, 2009; Morozkina et al, 2010) As such organisms need to live at thermal equilibrium with their environments, evolutionary pressure resulted in suitable adaptation of all cell components. A general model explaining protein thermal stability is still not available This limited understanding of structural, biophysical and evolutionary features related to thermostability or thermophilicity of proteins hampers the tuning of enzymes toward these properties. This is emphasized by the fact that random mutagenesis approaches (Asial et al, 2013; Stephens et al, 2014; Tian et al, 2014) still outperform rational protein design approaches in engineering a proteins’ thermal properties
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