Structural features determining thermal adaptation of esterases

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.

Keywords: esterase; molecular dynamics; psychrophilic, psychrotrophic, mesophilic, thermophilic bacteria; thermophilicity; thermostability.

Fig. 1

Structure-based sequence alignment of esterase Est2 from the thermophilic bacterium A. acidocaldarius with esterases EstB from mesophilic B. hailandensis, EstP from psychrotrophic Pseudomonas sp. B11-1, and EstS from psychrophilic S. halifaxensis. Notably, loops show lower sequence similarity when compared with the other regions. Sequences identical and similar in at least three residues were shaded in black and gray, respectively. The catalytic triad residues Ser, Asp and His are colored in yellow and the oxyanion hole residues in green. Residues colored in red follow the traffic rule reported for Est2 (Mandrich et al., 2009). Secondary structural elements of Est2 are shown above the alignment with α-helices as blue cylinders, β-strands as red arrows, and loops as gray lines, respectively (De Simone et al., 2000).

Fig. 2

SDS–PAGE analysis of purified recombinant esterases Est2, EstB, EstP and EstS. Enzymes carrying a His₆-tag at their C-terminal end were purified by the IMAC method, separated by SDS–PAGE on a 12% (w/v) polyacrylamide gel and stained with Coomassie Brilliant Blue G-250. Molecular weights of standard proteins are indicated on the left.

Fig. 3

Effect of temperature on activity and stability of esterases Est2, EstB, EstP and EstS. (A) The temperature optima were determined with p-nitrophenyl butyrate as the substrate at temperatures ranging from 5°C to 80°C. For comparison, Est2 values were divided by 10. Data are mean values of at least three independent measurements with standard deviations <10%. (B) The thermal stability of esterases Est2, EstB, EstP and EstS was determined after 1 h incubation at 5–80°C followed by measuring the residual activities with p-nitrophenyl butyrate as the substrate at 30°C. Esterase activity is expressed as percentage of the activity of samples before incubation. Data are mean values of at least three independent measurements with bars indicating the standard deviations.

Fig. 4

Structural comparison of esterases Est2, EstB, EstP and EstS. (A) Superimposition of the structure of Est2 (PDB ID: 1EVQ) with homology models of EstB, EstP and EstS. Secondary structural elements are labeled according to the structure of Est2. Loops with significant structural differences between the four structures are colored in black (Est2), orange (EstB), magenta (EstP) and green (EstS). For simplicity, structurally conserved loops are colored in gray and eight N-terminal amino acids of EstB that are not conserved among this group of enzymes are omitted. The catalytic residues Ser155, Asp252 and His282 of Est2 are shown as stick models in yellow. (B) Superimposition of the active sites of Est2, EstB, EstP und EstS. Orientations and distances of the catalytic triad residues are appropriate for establishing the H-bond network important for the catalytic activity of these esterases. Distances between catalytic Ser and His of Est2, EstB, EstP and EstS are 2.7, 3.8, 3.5 and 2.8 Å, respectively, and between His and Asp 3.2, 3.0, 3.1 and 2.7 Å. The HGGG motif containing two Gly residues forming the oxyanion hole close to the catalytic serine are indicated in orange. The catalytic triad residues are colored by element with carbon in gray, nitrogen in blue and oxygen in red; dotted lines represent H-bonds.

Fig. 5

MD simulations of EstS, EstP, EstB and Est2 movements at various temperatures. (A) MD simulations were performed for 100 ns and RMSF values for each residue were calculated for the last 30 ns of each simulation trajectory based on the fluctuations of the C_α atoms. Secondary structure elements, α-helices (blue rectangles) and β-strands (red arrows), are displayed along the sequences; flexible loops are labeled L2, L4, L10 and L12, respectively. (B) Comparison of RMSF values for overall enzyme structures and loop regions. Average RMSF of overall structures are represented by blue bars, and the further increment of the RMSF values for all loops in the proteins are indicated by green bars. Optimal temperatures for each protein are indicated by an asterisk above the bar. In A and B, results are shown for different esterases as indicated by the arrow on the right with blue color indicating optimal activity at lower and red at higher temperatures.

Fig. 6

Temperature-induced fluctuations of EstS, EstP, EstB and Est2. The red lines of the fluctuation plots indicate changes in RMSF between optimal temperature for enzyme catalysis (T_opt) and higher temperatures, T_high, for EstS, EstP, EstB and Est2 were 50°C, 50°C, 60°C and 80°C, respectively. The blue lines of the fluctuation plots indicate changes in RMSF between optimal temperature for enzyme catalysis (T_opt) and lower temperatures, T_low, for EstS, EstP, EstB and Est2 were 15°C, 15°C, 15°C and 50°C, respectively. Gray frames indicate the position of loops L2, L4, L10 and L12. Regions proposed to be related to protein stabilization and activity are indicated by and , respectively. Arrows at the right-hand side indicate an increase in inflexibility or rigidity.