title: Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-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 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 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.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA. xiichen@ucdavis.edu.
authors.forename: John B
authors.initials: JB
authors.lastname: McArthur
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Jie
authors.initials: J
authors.lastname: Zeng
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
mesh_terms: D003124:Colorimetry; D016296:Mutagenesis; D016979:Pasteurella multocida / Q000201:enzymology*; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013237:Stereoisomerism; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase