3S44

Crystal Structure of Pasteurella multocida sialyltransferase M144D mutant with CMP bound

3S44 の概要

• エントリーDOI: 10.2210/pdb3s44/pdb
• 関連するPDBエントリー: 2EX0 2EX1 2IHJ 2IHK 2IHZ 2ILV
• 分子名称: Alpha-2,3/2,6-sialyltransferase/sialidase, CMP-3F(a)-Neu5Ac (3 entities in total)
• 機能のキーワード: gt-b fold, sialyltransferase, transferase
• 由来する生物種: Pasteurella multocida
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 47039.79

構造登録者

Sugiarto, G.,Lau, K.,Li, Y.,Lim, S.,Ames, J.B.,Le, D.-T.,Fisher, A.J.,Chen, X. (登録日: 2011-05-18, 公開日: 2012-08-15, 最終更新日: 2023-09-13)

主引用文献

Sugiarto, G.,Lau, K.,Qu, J.,Li, Y.,Lim, S.,Mu, S.,Ames, J.B.,Fisher, A.J.,Chen, X.
A Sialyltransferase Mutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities for Directly Sialylating Lewis(x).
Acs Chem.Biol., 7:1232-1240, 2012

PubMed 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 Lewis(x) 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.

PubMed: 22583967
DOI: 10.1021/cb300125k

実験手法

X-RAY DIFFRACTION (1.45 Å)