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J. Biol. Chem., Vol. 278, Issue 14, 12157-12166, April 4, 2003
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From the Heparan sulfate
glycosaminoglycans are structurally complex polysaccharides
critically engaged in a wide range of cell and tissue functions. Any
structure-based approach to study their respective biological functions
is facilitated by the use of select heparan sulfate
glycosaminoglycan-degrading enzymes with unique substrate
specificities. We recently reported of one such enzyme, the
The Heparin/Heparan Sulfate 2-O-Sulfatase
from Flavobacterium heparinum
MOLECULAR CLONING, RECOMBINANT EXPRESSION, AND BIOCHEMICAL
CHARACTERIZATION*
§,¶,
,, and§§
Division of Biological Engineering,
Department of Biology, and Division
of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139 and ** Grampian
Enzymes, Ltd., Nisthouse Harray,
Orkney KW17 2LQ, United Kingdom
4,5-glycuronidase cloned from Flavobacterium heparinum
and recombinantly expressed in Escherichia coli (Myette,
J. R., Shriver, Z., Kiziltepe, T., McLean, M. W.,
Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry
41, 7424-7434). In this study, we likewise report the
molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active
enzyme. At the protein level, the flavobacterial
2-O-sulfatase possesses considerable sequence homology to
other members of a large sulfatase family, especially within its amino
terminus, where the highly conserved sulfatase domain is located.
Within this domain, we have identified by sequence homology the
critical active site cysteine predicted to be chemically modified as a formylglycine in vivo. We also present a characterization
of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of
its substrate specificity. In particular, we demonstrate that in
addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate. The
sulfatase also displays a clear kinetic preference for disaccharides
with 
1
4 linkages but is able, nevertheless, to hydrolyze
unsaturated, 2-O-sulfated chondroitin disaccharides.
Finally, we describe the substrate-product relationship of the
2-O-sulfatase to the 4,5-glycuronidase and the
analytical value of using both of these enzymes in tandem for
elucidating heparin/heparan sulfate composition.
*
This work was supported by National Institutes of Health
(NIH) Grants GM 57073 and CA90940.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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  O. Berteau, A. Guillot, A. Benjdia, and S. Rabot A New Type of Bacterial Sulfatase Reveals a Novel Maturation Pathway in Prokaryotes J. Biol. Chem., August 11, 2006; 281(32): 22464 - 22470. [Abstract] [Full Text] [PDF]
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R. Raman, J. R. Myette, Z. Shriver, K. Pojasek, G. Venkataraman, and R. Sasisekharan The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum. A STRUCTURAL AND BIOCHEMICAL STUDY OF THE ENZYME ACTIVE SITE AND SACCHARIDE SUBSTRATE SPECIFICITY J. Biol. Chem., March 28, 2003; 278(14): 12167 - 12174. [Abstract] [Full Text] [PDF]
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