Kinetics of Secoisolariciresinol Glucosyltransferase LuUGT74S1 and Its Mutants
Sadiq Saleh Moree, Lukas Böhm, Thomas Hoffmann, Wilfried G. Schwab
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.
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…
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Taxonomy
TopicsGlycosylation and Glycoproteins Research · Plant biochemistry and biosynthesis · Natural product bioactivities and synthesis