Thermoadaptation in an Ancestral Diterpene Cyclase by Altered Loop Stability

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Structural Biology

Background:

• Enzyme thermostability is critical for industrial applications like terpene cyclase-mediated synthesis of medicines and fine chemicals.
• Enhancing enzyme thermostability via traditional protein engineering can be challenging.
• Terpene cyclases, such as PtmT2, are vital in synthesizing complex molecules, including antibiotics.

Purpose of the Study:

• To create a hyperstable variant of the terpene cyclase PtmT2 using ancestral sequence reconstruction.
• To elucidate the molecular mechanisms behind increased thermostability and activity at elevated temperatures.
• To investigate the role of active site loop flexibility in enzyme thermoadaptation.

Main Methods:

• Ancestral sequence reconstruction to generate a hyperstable PtmT2 variant.
• Molecular dynamics simulations (μs timescale) to analyze protein behavior at different temperatures.
• Site-directed mutagenesis to probe the function of specific loop residues.

Main Results:

• An ancestral variant of PtmT2 exhibited a 40°C increase in melting temperature compared to the extant enzyme.
• Molecular dynamics revealed that a flexible active site loop maintains a productive conformation at high temperatures in the ancestral variant, unlike the extant enzyme.
• Specific loop mutations influenced both catalytic activity and thermostability, highlighting the loop's role in thermoadaptation.

Conclusions:

• Active site loop flexibility is a key determinant of thermoadaptation in terpene cyclases.
• Ancestral sequence reconstruction is a powerful tool for generating highly thermostable enzymes for industrial biotechnology.
• The hyperstable ancestral PtmT2 variant serves as a robust platform for further enzyme engineering.