Enzymes from psychrophiles display high catalytic efficiency at low temperatures. As a consequence, there is a lot of academic and industrial interest in investigating the molecular strategies adopted from these enzymes to work in conditions where other enzymes are almost inactive. Recently, a novel esterase activity was identified and isolated from the cold-adapted organism Pseudoalteromonas haloplanktis. The enzyme, named PhEST, is a dimer with a molecular mass of 60 kDa composed of two identical subunits. PhEST possesses four tryptophan residues that are homogenously dispersed in the protein tertiary organization. In this work, we used phosphorescence spectroscopy and molecular dynamics experiments to investigate the structural properties of PhEST. The obtained model structure of PhEST indicates that the environments of tryptophan residues W14 and W50 are characterized by limited conformational freedom. On the contrary, the environments of the tryptophan residues W181 and W197 are relatively mobile owing to enhanced fluctuations of residues 93-99 and 192-195, respectively, flexible loops that join segments of the protein secondary structure. The high-resolution phosphorescence spectrum in low-temperature glasses distinguishes two classes of Trp environments in PhEST structure: one class that is typical of compact internal hydrophobic sites, and the other class that is characteristic of disordered and/or partly solvent exposed regions. The phosphorescence lifetime of PhEST registered in fluid solution is invariably short, indicating that some Trp residues are in rather flexible superficial sites of the globular fold, whereas internal chromophores are strongly quenched by the proximity to Cys residues. Acrylamide and O2 quenching studies pointed out that the internal protein site is compact and rigid, typical of β-barrel core structures. Every spectroscopic feature described in this work is well accounted for by the proposed model structure of PhEST.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Materials Chemistry
- Surfaces, Coatings and Films