Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Koch Institute for Integrative Cancer Research, and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2009 Dec 11;284(50):35177-88. doi: 10.1074/jbc.M109.053801. Epub 2009 Sep 2.
Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.
肝素和硫酸乙酰肝素糖胺聚糖（HSGAG）组成了一类化学异质的硫酸多糖。为了研究这类多糖的结构-活性关系，需要鉴定和表征具有特定底物特异性和酶活性的降解酶。为此，我们在此报告了来自革兰氏阴性菌Flavobacterium heparinum 的 6-O-硫酸酯酶的分子克隆和广泛的结构功能分析。此外，我们还报告了该酶在大肠杆菌中的可溶性、活性形式的重组表达，并将其鉴定为特定的 HSGAG 硫酸酯酶。我们通过生化和结构研究进一步确定了酶的作用机制。通过使用定义明确的底物，我们研究了酶的动力学特性。通过同源建模研究对该分析进行了补充，该研究试图通过分析酶的活性位点拓扑结构，包括确定酶-底物相互作用的关键氨基酸并分配酶活性位点内的关键氨基酸，来合理化酶的底物特异性和作用模式。总之，我们的结构和生化研究表明，6-O-硫酸酯酶主要是一种外切酶，特异性作用于 HSGAG 寡糖底物非还原端存在的 N-硫酸化或 N-乙酰化 6-O-硫酸化的葡萄糖胺。这种对葡萄糖胺底物上的 N-乙酰基或 N-磺酸基的要求可以通过与酶活性位点内的关键残基产生有利的相互作用来解释。这些发现为使用 6-O-硫酸酯酶作为 HSGAG 结构-活性研究的工具提供了一个框架，并扩展了我们对这一类重要酶的生化和结构理解。
