Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Abstract

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Hotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase

“…According to the results of structure and sequence alignment analyses, the HSL family esterase (EstN7) from Bacillus cohnii strain N1 (PDB: 7B4Q, identity: 49.7%, RMSD: 1.067 Å) showed the highest structural identity with TrLipB, followed by the heroin esterase from Rhodococcus sp. strain H1 (PDB: 1LZK, identity: 47.1%, RMSD: 1.058 Å) and LipW from Mycobacterium marinum (PDB: 3QH4, identity: 40.9%, RMSD: 1.517 Å) .…”

“…Previously, we solved the crystal structure of TrLipB, which showed marked stability and resistance to organic solvents. Surprisingly, TrLipB shared high structural similarity with the HSL family esterase EstN7 from Bacillus cohnii strain N1 (PDB: 7B4Q, identity: 49.7%, RMSD 1.067 Å), an enzyme active at low temperatures . In this study, we aimed to understand the thermostable skeleton of TrLipB toward which we introduced substitutions on the rigid sites of the flexible region using both MD simulations and site-directed mutagenesis.…”

“…Surprisingly, TrLipB shared high structural similarity with the HSL family esterase EstN7 from Bacillus cohnii strain N1 (PDB: 7B4Q, identity: 49.7%, RMSD 1.067 Å), an enzyme active at low temperatures. 41 In this study, we aimed to understand the thermostable skeleton of TrLipB toward which we introduced substitutions on the rigid sites of the flexible region using both MD simulations and site-directed mutagenesis. This study might provide valuable insights into the rational design of the lipase thermostable skeleton and further industrial applications.…”

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability

“…Structure-based calculations can excel in detecting networks of rigid clusters through proteins and we have successfully used the FLEXOME implementation of pebble game rigidity analysis previously for this purpose 42,50 . We initially performed rigidity analysis on the available holo-HRP structure 19 .…”

“…A interaction with Phe152 in the non-glycosylated form could potentially disrupt the conformation of Phe41 relative to haem and thus may impact negatively on catalysis. Structure-based calculations can excel in detecting networks of rigid clusters through proteins and we have successfully used the FLEXOME implementation of pebble game rigidity analysis previously for this purpose 42,50 . We initially performed rigidity analysis on the available holo-HRP structure 19 .…”