Transesterification with CE15 glucuronoyl esterase from Cerrena unicolor reveals substrate preferences

Area of Science:

• Biochemistry
• Enzymology
• Lignin valorization

Background:

• Glucuronoyl esterases (GE, family CE15) are crucial for cleaving ester bonds in lignin-carbohydrate complexes (LCCs).
• Understanding GE substrate specificity, particularly alcohol preference, is key to predicting enzyme-lignin interactions and optimizing LCC degradation.
• The specific esterification positions (α-benzyl or γ-benzyl) preferred by fungal GEs remain largely uncharacterized.

Purpose of the Study:

• To investigate the alcohol preference of a fungal glucuronoyl esterase (CuGE) from Cerrena unicolor using transesterification reactions.
• To elucidate how CuGE's substrate preferences are influenced by the glucuronoyl donor and the alcohol acceptor molecule.

Main Methods:

• Enzyme-catalyzed transesterification reactions were employed using CuGE.
• A methyl ester of glucuronate or 4-O-methyl-glucuronate served as the donor substrate.
• Benzyl alcohol and 3-phenyl-1-propanol were used as acceptor molecules to assess alcohol preference.

Main Results:

• The fungal GE (CuGE) successfully catalyzed transesterification reactions, confirming its ability to form new ester bonds.
• CuGE demonstrated a clear preference for the γ-ester position when using the methyl ester of 4-O-methyl-glucuronate and 3-phenyl-1-propanol.
• The enzyme's substrate preference was primarily determined by the 4-O-methylation on the glucuronoyl donor, with a secondary influence from the type of alcohol acceptor.

Conclusions:

• The study successfully utilized transesterification to determine the alcohol preference of CuGE.
• CuGE exhibits specific substrate preferences, favoring 4-O-methylated glucuronoyl donors and certain alcohol acceptors.
• These findings provide insights into the enzymatic mechanisms involved in LCC modification and potential applications in biomass conversion.