# Kinetics of Secoisolariciresinol Glucosyltransferase LuUGT74S1 and Its Mutants

## TL;DR

This study identifies key amino acids in the enzyme LuUGT74S1 that affect its activity and the production of a more active compound, SMG.

## Contribution

The study reveals specific amino acid mutations that influence the enzyme's activity and SMG/SDG ratio, aiding biotechnological applications.

## Key findings

- Mutants S82F and E189L showed complete loss of enzymatic activity.
- A17S and Q136F mutants had the highest SMG/SDG ratios of 0.7 and 0.4.
- Diglucosylation is irreversible, while monoglycosylation is kinetically favored.

## Abstract

The lignan secoisolariciresinol
(SECO) diglucoside (SDG) is a phytoestrogen
with diverse effects. LuUGT74S1 glucosylates SECO to SDG, whereby
only small amounts of the monoglucoside SMG are formed intermediately,
which exhibit increased activity. To identify critical amino acids
that are important for enzymatic activity and the SMG/SDG ratio, 3D
structural modeling and docking, as well as site-directed mutation
studies, were performed. Enzyme assays with ten mutants revealed that
four of them had identical kinetic data to LuUGT74S1, while three
showed reduced and one increased catalytic efficiency kcat/Km. S82F and E189L substitutions
resulted in the complete absence of activity. A17 and Q136 are crucial
for the conversion of SMG to SDG as A17S and Q136F mutants exhibited
the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that
diglucosylation is an essentially irreversible reaction, while monoglycosylation
is kinetically favored. The results lay the foundation for the biotechnological
production of SMG.