Engineering Glycosyltransferase by Semi-Rational Design for Highly Regioselective Synthesis of Polydatin

Area of Science:

• Biochemistry
• Enzyme Engineering
• Metabolic Engineering

Background:

• Polydatin, a resveratrol glucoside, offers improved solubility and bioactivity.
• UDP-glucose-dependent glycosyltransferase UGT_BL1 from *Bacillus licheniformis* glycosylates resveratrol but lacks regioselectivity.
• Engineering regioselectivity is crucial for efficient polydatin synthesis.

Purpose of the Study:

• To enhance the regioselectivity of UGT_BL1 for polydatin production.
• To investigate the structural basis for regioselectivity through deletion modification.
• To analyze the impact of structural changes on enzyme-substrate interactions.

Main Methods:

• Deletion mutagenesis of UGT_BL1, specifically removing amino acids 62I and 63D to create the Δ(62-63) mutant.
• Enzymatic assays to determine polydatin regioselectivity.
• Molecular docking and dynamics simulations to analyze protein flexibility and active site interactions.

Main Results:

• The Δ(62-63) mutant exhibited a significant increase in polydatin regioselectivity, rising from 12.2% in the wild type to approximately 90.0%.
• Molecular simulations revealed that deleting amino acids 62I and 63D increased local flexibility.
• This enhanced flexibility facilitates the positioning of resveratrol's 3-OH group for targeted glycosylation.

Conclusions:

• Deletion modification of UGT_BL1 is an effective strategy to achieve high regioselectivity in polydatin synthesis.
• Structural flexibility in the enzyme's loop region plays a critical role in directing substrate glycosylation.
• This engineered enzyme offers a promising tool for the biotechnological production of polydatin.