A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Bacterial aryl sulfotransferases (AST) are enzymes that transfer sulfate groups.
• These enzymes typically use p-nitrophenylsulfate (pNPS) as a sulfuryl donor for phenolic acceptors.
• Growing interest exists due to their diverse acceptors and cost-effective donors, with ASTA previously shown to modify d-glucose.

Purpose of the Study:

• To develop and validate a directed evolution protocol for bacterial aryl sulfotransferase B (ASTB).
• To improve ASTB's ability to sulfurylate the carbohydrate N-acetylglucosamine (GlcNAc).
• To establish an efficient screening system for AST variants.

Main Methods:

• Advanced the pNPS quantification system for continuous screening in a 96-well microtiter plate (MTP) format.
• Created a random mutagenesis library (SeSaM) of ASTB and screened 1,760 clones.
• Utilized HPLC and MS analysis to confirm enzymatic activity and product formation.

Main Results:

• Developed a validated continuous screening system for AST with a coefficient of variation of 14.3%.
• Identified a beneficial variant, ASTB-V1 (Val579Asp), exhibiting up to a 3.4-fold increase in specific activity toward GlcNAc compared to wild-type ASTB (ASTB-WT).
• Confirmed a 2.4-fold increase in GlcNAc monosulfurylation product formation by ASTB-V1.

Conclusions:

• Successfully applied directed evolution to enhance ASTB for saccharide substrate modification.
• Demonstrated the first successful directed evolution of an AST for a saccharide substrate (GlcNAc).
• The developed screening system and ASTB-V1 variant offer a promising tool for carbohydrate sulfurylation.