title: A Photobacterium sp. α2-6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens.
abstract: In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2-6-sialyltransferase Psp2,6ST(15-501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15-501)-His6 toward α-GalNAc-containing acceptors by 21-115% without significantly affecting its sialylation activity to β-galactosides. Furthermore, the expression level was improved from 18-40 mg L(-1) for the wild-type enzyme to 72-110 mg L(-1) for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15-501)-His6 for synthesizing Neu5Acα2-6GalNAcαOSer/Thr STn antigens.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA; College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ding
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA; Department of Nutrition and Food Safety, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Go
authors.initials: G
authors.lastname: Sugiarto
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.affiliation: College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: wangjunru@nwsuaf.edu.cn.
authors.forename: Junru
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA. Electronic address: xiichen@ucdavis.edu.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
mesh_terms: D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003461:Crystallography; D008958:Models, Molecular; D009154:Mutation; D010776:Photobacterium / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C055491:sialosyl-Tn antigen; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
keywords: Improved protein expression; Mutagenesis; STn antigen; Sialyltransferase; Sialyltransferase mutant