Backbone and Methyl resonance assignment of an active PETase

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• LCC^ICCG is a bioengineered cutinase with high performance in degrading polyethylene terephthalate (PET).
• It shows enhanced thermal stability and PET hydrolysis activity over its predecessors and other PETases.
• Enzymatic degradation offers a promising route for PET recycling.

Purpose of the Study:

• To perform comprehensive resonance assignment of the polypeptide backbone and side chain methyl groups of the active LCC^ICCG enzyme.
• To provide residue-specific information for detailed structural and dynamic Nuclear Magnetic Resonance (NMR) analyses.
• To facilitate further understanding of LCC^ICCG's mechanism and optimize its application in PET recycling.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was employed for the study.
• Experiments were conducted at 60 °C utilizing a uniformly ¹⁵N-¹³C-labeled sample of LCC^ICCG.
• Resonance assignment covered both the polypeptide backbone and side chain methyl groups of the 27 kDa serine-hydrolase enzyme.

Main Results:

• Comprehensive resonance assignments for the backbone and methyl side chains of LCC^ICCG were successfully achieved.
• The assignments provide residue-specific insights into the enzyme's structure.
• The study leveraged the enzyme's thermostability, enabling experiments at elevated temperatures.

Conclusions:

• The detailed NMR assignments represent a significant advancement for structural and dynamic studies of LCC^ICCG.
• This work is a critical step towards a deeper understanding of the enzyme's function in PET degradation.
• The findings support LCC^ICCG's potential as a key enzyme for efficient and scalable PET recycling.