A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating Lewisx

Go Sugiarto, Kam Lau, Jingyao Qu, Yanhong Li, Sunghyuk Lim, Shengmao Mu, James B. Ames, Andrew J Fisher, Xi Chen

Research output: Contribution to journalArticle

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Abstract

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewisx antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

Original languageEnglish (US)
Pages (from-to)1232-1240
Number of pages9
JournalACS Chemical Biology
Volume7
Issue number7
DOIs
StatePublished - Jul 20 2012

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Sialyltransferases
Glycosyltransferases
Neuraminidase
Pasteurella multocida
Hydrolysis
Glycosylation
Substrates
Conformations
Catalysts
Glycoconjugates
Fucose
N-Acetylneuraminic Acid
Sugars
Nucleotides
Crystal structure
Carbohydrates
Nuclear magnetic resonance
X-Rays
Antigens
X rays

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

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A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating Lewisx . / Sugiarto, Go; Lau, Kam; Qu, Jingyao; Li, Yanhong; Lim, Sunghyuk; Mu, Shengmao; Ames, James B.; Fisher, Andrew J; Chen, Xi.

In: ACS Chemical Biology, Vol. 7, No. 7, 20.07.2012, p. 1232-1240.

Research output: Contribution to journalArticle

Sugiarto, Go ; Lau, Kam ; Qu, Jingyao ; Li, Yanhong ; Lim, Sunghyuk ; Mu, Shengmao ; Ames, James B. ; Fisher, Andrew J ; Chen, Xi. / A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating Lewisx In: ACS Chemical Biology. 2012 ; Vol. 7, No. 7. pp. 1232-1240.
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