Extending the enzymatic toolbox for heparosan polymerization, depolymerization, and detection

Area of Science:

• Biochemistry
• Microbiology
• Polymer Science

Background:

• Heparosan is an acidic polysaccharide crucial for heparan sulfate and heparin biosynthesis, showing high biocompatibility for pharmaceutical uses.
• Detecting heparosan in biological samples is challenging due to its low immunogenicity, hindering antibody development.

Purpose of the Study:

• To develop novel methods for controlled synthesis and depolymerization of heparosan.
• To enable the detection of heparosan in biological samples and facilitate pharmaceutical applications.

Main Methods:

• Utilized the enzyme repertoire of E. coli K5 and bacteriophage ΦK5B for heparosan synthesis and depolymerization.
• Employed fluorescently labeled heparosan nonamer to study the elongation mechanism of heparosan polymerases KfiA and KfiC.
• Identified and characterized the heparosan lyase activity of bacteriophage ΦK5B tailspike protein KflB, including its depolymerization mechanism.

Main Results:

• Demonstrated distributive action of polymerases KfiA and KfiC, producing labeled heparosan with low dispersity.
• Identified KflB as a heparosan lyase and characterized its endolytic depolymerization mechanism.
• Engineered an inactive KflB variant for sensitive heparosan detection in ELISA, blots, and cell-based assays.

Conclusions:

• Enzymatically synthesized heparosan serves as a valuable tool for biochemical studies and detection assays.
• The developed inactive KflB variant provides a robust method for heparosan detection, overcoming previous limitations.
• This work advances the utility of heparosan in pharmaceutical research and diagnostics.