Converting Pasteurella multocida alpha 2-3-sialyltransferase 1 (PmST1) to a regioselective alpha 2-6-sialyltransferase by saturation mutagenesis and regioselective screening

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida alpha 2-3-sialyltransferase 1 (PmST1) for alpha 2-6-sialyltransferase act...

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Bibliographic Details
Published in:Organic & biomolecular chemistry 2017-02, Vol.15 (7), p.1700-1709
Main Authors: McArthur, John B, Yu, Hai, Zeng, Jie, Chen, Xi
Format: Article
Language:English
Subjects:
Pasteurella multocida
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Summary:A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida alpha 2-3-sialyltransferase 1 (PmST1) for alpha 2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective alpha 2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.
ISSN:1477-0520
1477-0539
DOI:10.1039/c6ob02702d