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

doi:10.1016/j.carbpol.2023.121182

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

2023-11-01

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DOI：10.1016/j.carbpol.2023.121182

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Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e. g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunoge-nicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging.In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage & phi;K5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of & phi;K5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells.

Keywords:

Heparosan

Lyase

Glycosaminoglycans

Enzymatic synthesis

Bacteriophage

Tailspike protein

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

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