[Expression optimization and molecular modification of heparin C5 epimerase]

Area of Science:

• Biochemistry
• Enzymology
• Glycosaminoglycan Synthesis

Background:

• Heparin and heparan sulfate are crucial glycosaminoglycans used clinically as anticoagulants.
• Heparosan N-sulfate-glucuronate 5-epimerase (C5) is vital for synthesizing these compounds, catalyzing the conversion of D-glucuronic acid to L-iduronic acid.

Purpose of the Study:

• To optimize the expression and activity of zebrafish heparin C5 epimerase (Glce) in Escherichia coli.
• To engineer a more efficient C5 enzyme for potential applications in heparin biosynthesis.

Main Methods:

• Expressed the zebrafish Glce gene in E. coli using various vectors (pET-20b, pET-28a, pCold III).
• Utilized N-terminal fusion with the SET2 label to improve soluble protein expression.
• Performed site-directed mutagenesis near the substrate-binding pocket via rational design.

Main Results:

• The pCold III vector yielded the highest C5 activity (1873.61±5.42 U/L).
• SET2 fusion increased soluble expression by 50% and enzyme activity to (2409±6.43) U/L.
• The optimal V153R mutant showed significantly enhanced enzyme activity ((5804±5.63) U/L) and specific activity ((145.1±2.33) U/mg), representing 2.41-fold and 2.28-fold increases, respectively.

Conclusions:

• Optimization strategies, including vector selection, protein fusion, and rational mutagenesis, substantially improved heparin C5 epimerase expression and activity.
• These findings provide a foundation for the enzymatic, catalytic biosynthesis of heparin and related glycosaminoglycans.