1pnf
From Proteopedia
(New page: 200px<br /><applet load="1pnf" size="450" color="white" frame="true" align="right" spinBox="true" caption="1pnf, resolution 2.00Å" /> '''PNGASE F COMPLEX WIT...) |
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| - | [[Image:1pnf.gif|left|200px]]<br /><applet load="1pnf" size=" | + | [[Image:1pnf.gif|left|200px]]<br /><applet load="1pnf" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1pnf, resolution 2.00Å" /> | caption="1pnf, resolution 2.00Å" /> | ||
'''PNGASE F COMPLEX WITH DI-N-ACETYLCHITOBIOSE'''<br /> | '''PNGASE F COMPLEX WITH DI-N-ACETYLCHITOBIOSE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Crystallographic analysis and site-directed mutagenesis have been used to | + | Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile. |
==About this Structure== | ==About this Structure== | ||
| - | 1PNF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Elizabethkingia_meningoseptica Elizabethkingia meningoseptica] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine_amidase Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.52 3.5.1.52] Full crystallographic information is available from [http:// | + | 1PNF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Elizabethkingia_meningoseptica Elizabethkingia meningoseptica] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine_amidase Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.52 3.5.1.52] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1PNF OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Kuhn, P.]] | [[Category: Kuhn, P.]] | ||
| - | [[Category: Roey, P | + | [[Category: Roey, P Van.]] |
[[Category: SO4]] | [[Category: SO4]] | ||
[[Category: hydrolase]] | [[Category: hydrolase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:30:35 2008'' |
Revision as of 12:30, 21 February 2008
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PNGASE F COMPLEX WITH DI-N-ACETYLCHITOBIOSE
Overview
Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
About this Structure
1PNF is a Single protein structure of sequence from Elizabethkingia meningoseptica with as ligand. Active as Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase, with EC number 3.5.1.52 Full crystallographic information is available from OCA.
Reference
Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F., Kuhn P, Guan C, Cui T, Tarentino AL, Plummer TH Jr, Van Roey P, J Biol Chem. 1995 Dec 8;270(49):29493-7. PMID:7493989
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