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PDBsum entry 1hm2
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References listed in PDB file
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Key reference
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Title
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Active site of chondroitin ac lyase revealed by the structure of enzyme-Oligosaccharide complexes and mutagenesis.
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Authors
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W.Huang,
L.Boju,
L.Tkalec,
H.Su,
H.O.Yang,
N.S.Gunay,
R.J.Linhardt,
Y.S.Kim,
A.Matte,
M.Cygler.
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Ref.
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Biochemistry, 2001,
40,
2359-2372.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structures of Flavobacterium heparinium chondroitin AC lyase
(chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide
(DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide
(HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively.
The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate
tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For
each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and
HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC
DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1,
and +2, within a narrow and shallow protein channel. We suggest that subsites -2
and -1 together represent the substrate recognition area, +1 is the catalytic
subsite and +1 and +2 together represent the product release area. The putative
catalytic site is located between the substrate recognition area and the product
release area, carrying out catalysis at the +1 subsite. Four residues near the
catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic
tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed
residual activity for only the Arg292Ala mutant. Structural data indicate that
Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties
of galactosamine, but does not participate directly in catalysis. Candidates for
the general base, removing the proton attached to C-5 of the glucuronic acid at
the +1 subsite, are Tyr234, which could be transiently deprotonated during
catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving
group. Arginine 288 likely contributes to charge neutralization and
stabilization of the enolate anion intermediate during catalysis.
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