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Sugar binding protein, lyase
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PDB id
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3e7j
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Contents |
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* Residue conservation analysis
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DOI no:
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J Biol Chem
285:20051-20061
(2010)
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PubMed id:
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Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.
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D.Shaya,
W.Zhao,
M.L.Garron,
Z.Xiao,
Q.Cui,
Z.Zhang,
T.Sulea,
R.J.Linhardt,
M.Cygler.
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ABSTRACT
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Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both
heparin and heparan sulfate glycosaminoglycans through a beta-elimination
mechanism. Recently, we determined the crystal structure of HepII from
Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex
with a heparin disaccharide product, and identified the location of its active
site. Here we present the structure of HepII complexed with a heparan sulfate
disaccharide product, proving that the same binding/active site is responsible
for the degradation of both uronic acid epimers containing substrates. The key
enzymatic step involves removal of a proton from the C5 carbon (a chiral center)
of the uronic acid, posing a topological challenge to abstract the proton from
either side of the ring in a single active site. We have identified three
potential active site residues equidistant from C5 and located on both sides of
the uronate product and determined their role in catalysis using a set of
defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for
both substrates and we determined its crystal structure complexed with a heparan
sulfate-derived tetrasaccharide. Based on kinetic characterization of various
mutants and the structure of the enzyme-substrate complex we propose residues
participating in catalysis and their specific roles.
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