Impact of phenylalanines outside the dimer interface on phosphotriesterase stability and function

Area of Science:

• Biochemistry
• Protein Engineering
• Computational Biology

Background:

• Previous studies indicated phenylalanine's role in phosphotriesterase (PTE) dimer stabilization via para-fluorophenylalanine (pFF) incorporation.
• pFF incorporation improved PTE stability but decreased solubility due to non-specific integration.
• Investigating solvent-exposed phenylalanines offers a strategy to enhance PTE without compromising solubility.

Purpose of the Study:

• To explore the function and stability roles of solvent-exposed phenylalanine residues in phosphotriesterase (PTE) outside the dimer interface.
• To identify specific phenylalanine residues critical for PTE activity and stability.
• To evaluate computational predictions against experimental mutagenesis data.

Main Methods:

• Site-directed mutagenesis was used to create eight single solvent-exposed phenylalanine mutants of PTE.
• Computational modeling (Rosetta) was employed to predict the effects of these mutations.
• Enzyme activity assays and stability assessments were performed on the generated mutants.
• Structural integrity was evaluated for key variants.

Main Results:

• Computational predictions showed good agreement with experimental observations.
• Three residues (F304, F327, F335) were identified as critical for PTE activity and stability, despite not being at the dimer interface or active site.
• The F306L variant demonstrated improved enzymatic activity at both ambient and elevated temperatures.
• Other phenylalanine mutants showed no significant impact on PTE structure or activity.

Conclusions:

• Solvent-exposed phenylalanine residues play significant roles in PTE function and stability beyond the dimer interface.
• Specific residues like F304, F327, and F335 are key modulators of PTE performance.
• The F306L mutation presents a promising avenue for developing PTE variants with enhanced thermal stability and activity.