2e9l: Difference between revisions
New page: left|200px<br /> <applet load="2e9l" size="450" color="white" frame="true" align="right" spinBox="true" caption="2e9l, resolution 1.60Å" /> '''Crystal Structure o... |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:2e9l.gif|left|200px]]<br /> | [[Image:2e9l.gif|left|200px]]<br /><applet load="2e9l" size="350" color="white" frame="true" align="right" spinBox="true" | ||
<applet load="2e9l" size=" | |||
caption="2e9l, resolution 1.60Å" /> | caption="2e9l, resolution 1.60Å" /> | ||
'''Crystal Structure of human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Prote:KLrP) complex with Glucose and fatty acids'''<br /> | '''Crystal Structure of human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Prote:KLrP) complex with Glucose and fatty acids'''<br /> | ||
| Line 6: | Line 5: | ||
==Overview== | ==Overview== | ||
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the, activity of a conduritol B epoxide (CBE)-insensitive neutral, glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and, rat brains and human fibroblasts. The candidates for the enzyme were, assigned to the Klotho (KL) whose family members share a -glucosidase-like, domain but whose natural substrates unknown. Among this family, only the, KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when, expressed in CHOP cells, in which Myc-tagged KLrP was exclusively, distributed in the cytosol. In addition, knockdown of the endogenous KLrP, by siRNA increased the cellular level of GlcCer. The purified recombinant, KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic, GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl, derivatives but each kcat/Km was much lower than that for glucosyl, derivatives. The X-ray structure of KLrP at 1.6 resolution revealed that, KLrP is a (/)8 TIM barrel, in which E165 and E373 at the carboxyl termini, of -strands 4 and 7 could function as an acid/base catalyst and, nucleophile, respectively. The distance between carboxyl oxygens of these, two residues is 5.3 indicating the reaction proceeds with the anomeric, carbon retained upon cleavage, rather than inverted. The substrate-binding, cleft of the enzyme was occupied with palmitic acid and oleic acid when, the recombinant protein was crystallized in a complex with glucose. GlcCer, was found to well fit the cleft of the crystal structure of KLrP., Collectively, KLrP was identified as a cytosolic neutral, glycosylceramidase which could be involved in a novel non-lysosomal, catabolic pathway of GlcCer. | Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the, activity of a conduritol B epoxide (CBE)-insensitive neutral, glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and, rat brains and human fibroblasts. The candidates for the enzyme were, assigned to the Klotho (KL) whose family members share a -glucosidase-like, domain but whose natural substrates unknown. Among this family, only the, KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when, expressed in CHOP cells, in which Myc-tagged KLrP was exclusively, distributed in the cytosol. In addition, knockdown of the endogenous KLrP, by siRNA increased the cellular level of GlcCer. The purified recombinant, KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic, GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl, derivatives but each kcat/Km was much lower than that for glucosyl, derivatives. The X-ray structure of KLrP at 1.6 resolution revealed that, KLrP is a (/)8 TIM barrel, in which E165 and E373 at the carboxyl termini, of -strands 4 and 7 could function as an acid/base catalyst and, nucleophile, respectively. The distance between carboxyl oxygens of these, two residues is 5.3 indicating the reaction proceeds with the anomeric, carbon retained upon cleavage, rather than inverted. The substrate-binding, cleft of the enzyme was occupied with palmitic acid and oleic acid when, the recombinant protein was crystallized in a complex with glucose. GlcCer, was found to well fit the cleft of the crystal structure of KLrP., Collectively, KLrP was identified as a cytosolic neutral, glycosylceramidase which could be involved in a novel non-lysosomal, catabolic pathway of GlcCer. | ||
==About this Structure== | ==About this Structure== | ||
2E9L is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with BGC, PLM, OLA and GOL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-glucosidase Beta-glucosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.21 3.2.1.21] Full crystallographic information is available from [http:// | 2E9L is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=BGC:'>BGC</scene>, <scene name='pdbligand=PLM:'>PLM</scene>, <scene name='pdbligand=OLA:'>OLA</scene> and <scene name='pdbligand=GOL:'>GOL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Beta-glucosidase Beta-glucosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.21 3.2.1.21] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2E9L OCA]. | ||
==Reference== | ==Reference== | ||
| Line 29: | Line 25: | ||
[[Category: novel cytosolic neutral beta-glycosylceramidase]] | [[Category: novel cytosolic neutral beta-glycosylceramidase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jan 23 13:38:05 2008'' | ||
Revision as of 14:38, 23 January 2008
|
Crystal Structure of human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Prote:KLrP) complex with Glucose and fatty acids
OverviewOverview
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the, activity of a conduritol B epoxide (CBE)-insensitive neutral, glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and, rat brains and human fibroblasts. The candidates for the enzyme were, assigned to the Klotho (KL) whose family members share a -glucosidase-like, domain but whose natural substrates unknown. Among this family, only the, KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when, expressed in CHOP cells, in which Myc-tagged KLrP was exclusively, distributed in the cytosol. In addition, knockdown of the endogenous KLrP, by siRNA increased the cellular level of GlcCer. The purified recombinant, KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic, GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl, derivatives but each kcat/Km was much lower than that for glucosyl, derivatives. The X-ray structure of KLrP at 1.6 resolution revealed that, KLrP is a (/)8 TIM barrel, in which E165 and E373 at the carboxyl termini, of -strands 4 and 7 could function as an acid/base catalyst and, nucleophile, respectively. The distance between carboxyl oxygens of these, two residues is 5.3 indicating the reaction proceeds with the anomeric, carbon retained upon cleavage, rather than inverted. The substrate-binding, cleft of the enzyme was occupied with palmitic acid and oleic acid when, the recombinant protein was crystallized in a complex with glucose. GlcCer, was found to well fit the cleft of the crystal structure of KLrP., Collectively, KLrP was identified as a cytosolic neutral, glycosylceramidase which could be involved in a novel non-lysosomal, catabolic pathway of GlcCer.
About this StructureAbout this Structure
2E9L is a Single protein structure of sequence from Homo sapiens with , , and as ligands. Active as Beta-glucosidase, with EC number 3.2.1.21 Full crystallographic information is available from OCA.
ReferenceReference
Klotho-related protein is a novel cytosolic neutral beta -glycosylceramidase., Hayashi Y, Okino N, Kakuta Y, Shiknai T, Tani M, Narimatsu H, Ito M, J Biol Chem. 2007 Jun 26;. PMID:17595169
Page seeded by OCA on Wed Jan 23 13:38:05 2008