1m01: Difference between revisions

Revision as of 14:50, 21 February 2008

Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)

OverviewOverview

SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site of SpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.

About this StructureAbout this Structure

1M01 is a Single protein structure of sequence from Streptomyces plicatus with , , and as ligands. Active as Beta-N-acetylhexosaminidase, with EC number 3.2.1.52 Full crystallographic information is available from OCA.

ReferenceReference

Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state., Williams SJ, Mark BL, Vocadlo DJ, James MN, Withers SG, J Biol Chem. 2002 Oct 18;277(42):40055-65. Epub 2002 Aug 8. PMID:12171933

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OCA

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-[[Image:1m01.gif|left|200px]]<br /><applet load="1m01" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1m01.gif|left|200px]]<br /><applet load="1m01" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1m01, resolution 2.10&Aring;" />
 '''Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)'''<br />
 ==Overview==
-SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating, evidence suggests that the hydrolytic mechanism involves, substrate-assisted catalysis wherein the 2-acetamido substituent acts as a, nucleophile to form an oxazolinium ion intermediate. The role of a, conserved aspartate residue (D313) in the active site of SpHex was, investigated through kinetic and structural analyses of two variant, enzymes, D313A and D313N. Three-dimensional structures of the wild-type, and variant enzymes in product complexes with N-acetyl-d-glucosamine, revealed substantial differences. In the D313A variant the 2-acetamido, group was found in two conformations of which only one is able to aid in, catalysis through anchimeric assistance. The mutation D313N results in a, steric clash in the active site between Asn-313 and the 2-acetamido group, preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the, insensitivity of this variant to chemical rescue. By comparison, the D313A, mutation results in a shift in a shift in the pH optimum and a modest, decrease in activity that can be rescued by using azide as an exogenous, nucleophile. These structural and kinetic data provide evidence that, Asp-313 stabilizes the transition states flanking the oxazoline, intermediate and also assists to correctly orient the 2-acetamido group, for catalysis. Based on analogous conserved residues in the family 18, chitinases and family 56 hyaluronidases, the roles played by the Asp-313, residue is likely general for all hexosaminidases using a mechanism, involving substrate-assisted catalysis.
+SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site of SpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.
 ==About this Structure==
-M01 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_plicatus Streptomyces plicatus] with NAG, CL, SO4 and GOL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-N-acetylhexosaminidase Beta-N-acetylhexosaminidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.52 3.2.1.52] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1M01 OCA].
+M01 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_plicatus Streptomyces plicatus] with <scene name='pdbligand=NAG:'>NAG</scene>, <scene name='pdbligand=CL:'>CL</scene>, <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=GOL:'>GOL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-N-acetylhexosaminidase Beta-N-acetylhexosaminidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.52 3.2.1.52] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1M01 OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Streptomyces plicatus]]
-[[Category: James, M.N.G.]]
+[[Category: James, M N.G.]]
-[[Category: Mark, B.L.]]
+[[Category: Mark, B L.]]
-[[Category: Vocadlo, D.J.]]
+[[Category: Vocadlo, D J.]]
-[[Category: Williams, S.J.]]
+[[Category: Williams, S J.]]
-[[Category: Withers, S.G.]]
+[[Category: Withers, S G.]]
 [[Category: CL]]
 [[Category: GOL]]
@@ Line 28: / Line 28: @@
 [[Category: tim barrel]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Sun Nov 25 03:34:55 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:50:03 2008''