Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum

Abstract

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.

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum

“…Bacterial Expression and Protein Purification-Similar to the setup in the 2-O-sulfatase (18) and 6-O-sulfatase (19) expression systems that we had designed earlier, the Escherichia coli strain BL21(DE3) and one-step affinity purification by nickel chelation chromatography likewise were used. von Hejne computational method was used to make the prediction of the NH 2 -terminal signal sequence and putative cleavage site for the protein (25).…”

“…Molecular Cloning of Flavobacterial N-Sulfamidase-PCR from a ZAPII flavobacterial genomic library originally screened using DNA hybridization probes specific to the 2-Osulfatase (18) and subsequently used for the 6-O-sulfatase (19) were used to clone the sulfamidase gene. Library construction, hybridization screening, and phage excision were performed as outlined.…”

“…The coding sequence of orfc was identified by the canonical Protein Families Database (24) sulfatase family identifier (CXPXRXXXX(S/T)G) and subsequently PCR-amplified using the primer set given by 5Ј-TCT AGA CAT ATG AAA TTT AAC AAA TTG AAA TAT TTC-3Ј (forward) and 5Ј-GGA TCC TCG AGT TAC TTC AAA TAA TTG TAA CTG GAA T-3Ј (reverse). Subcloning of the amplified gene into T7-based bacterial expression vector pET28a (Novagen) was performed as an NdeI-XhoI cassettes described for the 6-O-sulfatase (19).…”

“…Finally, taken together with the reported substrate specificities for the previously characterized F. heparinum 2-O-sulfatase, the 6-O-sulfatase (described in the preceding paper, see Ref. 19) and the unsaturated glucuronyl hydrolase, we are now able to reconstruct in vitro the complete and 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 HSGAG-derived oligosaccharides.…”

Structural Determinants in Streptococcal Unsaturated Glucuronyl Hydrolase for Recognition of Glycosaminoglycan Sulfate Groups

“…Glycosaminoglycan lyases and UGL are also prerequisites for cell growth in these bacteria. Although the motif for degradation of sulfated glycosaminoglycans is not induced in F. heparinum UGL, this bacterium produces some sulfatases involved in desulfation of glycosaminoglycans (42,43).…”