Investigations on β1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate
Investigations on β1,4-galactosyltransferase I using 6-sulfo-GlcNAc as an acceptor sugar substrate
复制标题
使用 6-sulfo-GlcNAc 作为受体糖底物研究 β1,4-半乳糖基转移酶 I
DOI：
10.1007/s10719-013-9488-4
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发表时间：
2013
影响因子：
3
通讯作者：
P. Qasba
中科院分区：
文献类型：
--
作者：
B. Ramakrishnan;Anthony J. Moncrief;Tyler A. Davis;L. Holland;P. Qasba
6-硫酸化修饰N-乙酰葡糖胺（6-磺基-GlcNAc）经常被发现是许多生物学上重要的碳水化合物表位如6-磺基-LeX的一部分。在这些表位中，6-磺基-GlcNAc部分通过β1-4键中的半乳糖延伸。在锰存在下，β 4GalT 1酶将半乳糖（Gal）从UDP-Gal转移至N-乙酰葡糖胺（GlcNAc）。在此，我们报告了β 4GalT 1酶将Gal转移至6-磺基-GlcNAc和4-甲基伞形酮基-6-磺基-N-乙酰基-β-D-氨基葡萄糖苷（6-磺基-β GlcNAc-MU）受体底物，尽管效率非常低。为了了解GlcNAc受体上的6-硫酸基团对β 4GalT 1分子的催化活性的影响，我们测定了牛β 4GalT 1突变酶M344 H-β 4GalT 1与6-磺基-GlcNAc分子复合物的催化结构域的晶体结构。在晶体结构中，6-磺基-GlcNAc以类似于GlcNAc分子的方式与蛋白质结合。然而，6-硫酸基团与蛋白质分子的疏水区残基276-285进行额外的相互作用，并且发现该基团楔入Phe-280和Trp 314残基的芳香族侧链之间。由于Trp 314残基的侧链在酶的催化循环期间经历构象变化，Trp 314和6-硫酸基团之间的分子相互作用可能阻碍这种构象变化。因此，缺乏有利的结合环境，加上阻碍构象变化，可能是导致催化活性差的原因。
6-sulfate modified N-acetylglucosamine (6-sulfo-GlcNAc) is often found as part of many biologically important carbohydrate epitopes such as 6-sulfo-LeX. In these epitopes, the 6-sulfo-GlcNAc moiety is extended by a galactose sugar in a β1-4 linkage. The β4GalT1 enzyme transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) in the presence of manganese. Here we report that the β4GalT1 enzyme transfers Gal to the 6-sulfo-GlcNAc and 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide (6-sulfo-βGlcNAc-MU) acceptor substrates, although with very low efficiency. To understand the effect that the 6-sulfate group on the GlcNAc acceptor has on the catalytic activity of the β4GalT1 molecule, we have determined the crystal structure of the catalytic domain of bovine β4GalT1 mutant enzyme M344H-β4GalT1 complex with the 6-sulfo-GlcNAc molecule. In the crystal structure, the 6-sulfo-GlcNAc is bound to the protein in a way that is similar to the GlcNAc molecule. However, the 6-sulfate group engages in additional interactions with the hydrophobic region, residues 276–285, of the protein molecule, and this group is found wedged between the aromatic side chains of Phe-280 and Trp314 residues. Since the side chain of the Trp314 residue undergoes conformational changes during the catalytic cycle of the enzyme, molecular interaction between Trp314 and the 6-sulfate group might hinder this conformational change. Therefore, the lack of a favorable binding environment, together with hindrance to the conformational changes, might be responsible for the poor catalytic activity.
影响因子：
3.2
作者：
P. Qasba;B. Ramakrishnan;E. Boeggeman
通讯作者：
P. Qasba;B. Ramakrishnan;E. Boeggeman
影响因子：
4.8
作者：
Ramakrishnan, B;Shah, PS;Qasba, PK
通讯作者：
Qasba, PK
影响因子：
5.6
作者：
Ramakrishnan, B;Qasba, PK
通讯作者：
Qasba, PK