Increasing the Stability of Flavin-Dependent Halogenases by Disulfide Engineering

Area of Science:

• Biochemistry
• Enzyme Engineering
• Organic Chemistry

Background:

• Flavin-dependent halogenases catalyze regioselective halogenation of aromatic compounds under mild conditions.
• Improving enzyme stability and efficiency is crucial for expanding halogenase applications.
• Thermostable variants of tryptophan 6-halogenase (Thal) exhibit dimerization, unlike the wild type.

Purpose of the Study:

• To enhance the stability and efficiency of flavin-dependent halogenases.
• To investigate the effect of covalent dimerization on enzyme stability.
• To explore the transferability of stabilization strategies to other halogenases.

Main Methods:

• Engineering a covalently dimerized Thal variant (Thal CC) by introducing cysteine residues at the dimer interface.
• Assessing the thermostability and long-term stability of Thal CC compared to wild-type Thal.
• Introducing homologous mutations into tryptophan 5-halogenase (PyrH) to evaluate transferability and further optimize stability.

Main Results:

• The Thal CC variant showed significantly increased thermostability (ΔT50 = 15.7 K) and improved stability over time at elevated temperatures.
• Covalent dimerization successfully stabilized Thal and was transferable to PyrH.
• Further stabilization of PyrH was achieved by introducing cysteine mutations at alternative dimer interface sites.

Conclusions:

• Covalent dimerization is an effective strategy for enhancing flavin-dependent halogenase stability.
• This stabilization approach can be applied to different halogenase enzymes, such as PyrH.
• Further optimization of dimer interfaces can lead to even greater thermostability in engineered halogenases.