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

ABSTRACT: 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?2-3-sialyltransferase 1 (PmST1) for ?2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective ?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.

SUBMITTER: McArthur JB

PROVIDER: S-EPMC5333693 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

McArthur John B JB Yu Hai H Zeng Jie J Chen Xi X

Organic & biomolecular chemistry 20170130 7

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 multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activ ...[more]

PMID: 28134951

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