The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (14), p.12167-12174
• Main Authors: Raman, Rahul, Myette, James R, Shriver, Zachary, Pojasek, Kevin, Venkataraman, Ganesh, Sasisekharan, Ram
• Format: Article
• Language: English
• Subjects: Alanine - analogs & derivatives; Binding Sites - genetics; Disaccharides - metabolism; Flavobacterium - enzymology; Glycine - analogs & derivatives; Heparin - metabolism; Heparitin Sulfate - metabolism; Metals - chemistry; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Mapping; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Sulfatases - chemistry; Sulfatases - genetics; Sulfatases - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M211425200

In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.