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Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan

The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modificati...

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Detalles Bibliográficos
Autores principales: Xi, Xintong, Hu, Litao, Huang, Hao, Wang, Yang, Xu, Ruirui, Du, Guocheng, Chen, Jian, Kang, Zhen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10291996/
https://www.ncbi.nlm.nih.gov/pubmed/37327079
http://dx.doi.org/10.1093/jimb/kuad012
Descripción
Sumario:The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies, including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment, and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP–hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37°C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.