title: Heparin/heparan sulfate N-sulfamidase from Flavobacterium heparinum: structural and biochemical investigation of catalytic nitrogen-sulfur bond cleavage.
abstract: 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.
authors.affiliation: 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.
authors.forename: James R
authors.initials: JR
authors.lastname: Myette
authors.forename: Venkataramanan
authors.initials: V
authors.lastname: Soundararajan
authors.forename: Jonathan
authors.initials: J
authors.lastname: Behr
authors.forename: Zachary
authors.initials: Z
authors.lastname: Shriver
authors.forename: Rahul
authors.initials: R
authors.lastname: Raman
authors.forename: Ram
authors.initials: R
authors.lastname: Sasisekharan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005417:Flavobacterium / Q000201:enzymology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002118:Calcium; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D006867:Hydrolases; D009584:Nitrogen; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013455:Sulfur; C022708:N-sulfoglucosamine sulfohydrolase