Engineering ribonucleoside triphosphate specificity in a thymidylyltransferase.

Area of Science:

• Biochemistry
• Enzymology
• Synthetic Biology

Background:

• Glycosyltransferases are crucial biological catalysts involved in numerous cellular processes.
• Chemical synthesis of sugar nucleotides is challenging, limiting research on glycosyltransferases.
• Enzymatic synthesis offers an alternative but is constrained by enzyme substrate specificity.

Purpose of the Study:

• To engineer the promiscuous thymidylyltransferase Cps2L from Streptococcus pneumoniae.
• To enhance its uridylyltransferase and guanidyltransferase activities for broader sugar nucleotide production.
• To explore the potential of the engineered enzyme in synthesizing novel nucleotide sugars.

Main Methods:

• Active site engineering of Cps2L via site-directed mutagenesis at position Q24.
• Enzymatic assays to quantify the production of various UDP- and GDP-sugars.
• Evaluation of the variant enzyme's catalytic efficiency with different sugar phosphate substrates.

Main Results:

• The Q24S variant showed 10-, 3-, and 2-fold enhancements in UDP-glucosamine, UDP-mannose, and UDP-N-acetylglucosamine production, respectively.
• The variant exhibited novel catalytic activity, including GDP-mannose formation.
• Significant yields of dTDP-Galactose furanose (90%) and UDP-Arabinose furanose (30%) were achieved, along with the synthesis of UDP-3-O-alkylglucose derivatives.

Conclusions:

• Active site engineering of Cps2L at Q24 significantly broadens its nucleotidylyltransferase activity.
• The Q24S variant demonstrates enhanced production of various nucleotide sugars and novel catalytic functions.
• This engineered enzyme represents a promising new approach for the enzymatic synthesis of diverse nucleotide sugars.