Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity.
Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity.
复制标题
DOI：
10.1021/bi012147o
复制
发表时间：
2002-05
期刊：
影响因子：
2.9
通讯作者：
James R. Myette;Z. Shriver;T. Kiziltepe;M. W. McLean;G. Venkataraman;R. Sasisekharan
中科院分区：
文献类型：
--
作者：
James R. Myette;Z. Shriver;T. Kiziltepe;M. W. McLean;G. Venkataraman;R. Sasisekharan
土壤细菌肝黄杆菌产生几种酶，以序列特异性的方式降解硫酸肝素糖胺聚糖（HSGAGs）。其中，这些酶包括肝素酶和一种不寻常的糖醛酸酶，它能水解低聚糖非还原端由肝素酶消除性裂解产生的不饱和delta4,5醛酸。本文报道了黄杆菌基因组中delta4,5糖醛酸酶基因的克隆及其在大肠杆菌中的重组表达。我们还报道了该酶的生化和动力学表征，包括对其底物特异性的分析。我们发现delta4,5糖醛酸酶根据糖苷键和其糖底物内的硫酸化模式进行区分。特别是，我们发现糖醛酸酶对1- > - 4键表现出强烈的偏好，使这种酶特异性地针对肝素/硫酸肝素而不是1- > - 3键的糖胺聚糖，如软骨素/硫酸皮肤素或透明质酸。最后，我们证明了这种酶在肝素酶衍生的HSGAG低聚糖测序中的应用。
The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.