Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF CATALYTIC NITROGEN-SULFUR BOND CLEAVAGE
Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF CATALYTIC NITROGEN-SULFUR BOND CLEAVAGE
复制标题
DOI：
10.1074/jbc.m109.053835
复制
发表时间：
2009-12-11
影响因子：
4.8
通讯作者：
Sasisekharan, Ram
中科院分区：
文献类型：
--
作者：
Myette, James R.;Soundararajan, Venkataramanan;Sasisekharan, Ram
硫酸化多糖如肝素和硫酸乙酰肝素糖胺聚糖（HSGAG）是化学和结构异质的生物聚合物，其作为许多生物功能的关键调节剂起作用。HSGAG精细结构的阐明是理解其功能多样性的基础，并且这通过使用具有限定底物特异性的选择降解酶来促进。我们以前的研究已经报道了在大肠杆菌中的肝素黄杆菌2-O-硫酸酯酶和6-O-硫酸酯酶的克隆、表征、重组表达和结构-功能分析，所述肝素黄杆菌2-O-硫酸酯酶和6-O-硫酸酯酶从HSGAG聚合物的特定位置切割O-硫酸酯基团。基于这些先前的研究，我们在这里报告的分子克隆和重组表达在大肠杆菌中的N-磺酰胺酶，特异性HSGAGs。此外，我们通过分子模拟研究和底物特异性和基本生物化学的结构-功能分析来研究这种酶的基本酶学。我们使用这些研究的结果，提出了一种新的机制，氮-硫键裂解的N-磺酰胺酶。总之，我们的结构和生物化学研究表明，N-磺酰胺酶是一种主要的胞外酶，特异性作用于作为单糖或奇数寡糖底物的非还原端存在的N-硫酸化和6-O-葡糖胺。结合先前报道的F.肝素2-O-硫酸酯酶、6-O-硫酸酯酶和不饱和葡萄糖醛酸水解酶，我们现在能够在体外重建F.肝素，并将这些酶串联应用于肝素衍生的寡糖的外切测序。
Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.