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1m01
From Proteopedia
(New page: 200px<br /><applet load="1m01" size="450" color="white" frame="true" align="right" spinBox="true" caption="1m01, resolution 2.10Å" /> '''Wildtype Streptomyce...) |
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| - | [[Image:1m01.gif|left|200px]]<br /><applet load="1m01" size=" | + | [[Image:1m01.gif|left|200px]]<br /><applet load="1m01" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1m01, resolution 2.10Å" /> | caption="1m01, resolution 2.10Å" /> | ||
'''Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)'''<br /> | '''Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)'''<br /> | ||
==Overview== | ==Overview== | ||
| - | SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-beta-hexosaminides. Accumulating | + | 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== | ==About this Structure== | ||
| - | 1M01 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:// | + | 1M01 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== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Streptomyces plicatus]] | [[Category: Streptomyces plicatus]] | ||
| - | [[Category: James, M | + | [[Category: James, M N.G.]] |
| - | [[Category: Mark, B | + | [[Category: Mark, B L.]] |
| - | [[Category: Vocadlo, D | + | [[Category: Vocadlo, D J.]] |
| - | [[Category: Williams, S | + | [[Category: Williams, S J.]] |
| - | [[Category: Withers, S | + | [[Category: Withers, S G.]] |
[[Category: CL]] | [[Category: CL]] | ||
[[Category: GOL]] | [[Category: GOL]] | ||
| Line 28: | Line 28: | ||
[[Category: tim barrel]] | [[Category: tim barrel]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:50:03 2008'' |
Revision as of 11:50, 21 February 2008
|
Wildtype Streptomyces plicatus beta-hexosaminidase in complex with product (GlcNAc)
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.
About 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.
Reference
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
Page seeded by OCA on Thu Feb 21 13:50:03 2008
