2e9l: Difference between revisions
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==Overview== | ==Overview== | ||
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the | Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-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 beta-glucosidase-like domain but whose natural substrates are 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 small interfering RNA 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 k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. 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 fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer. | ||
==Disease== | |||
Known disease associated with this structure: Corticosteroid-binding globulin deficiency OMIM:[[http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=122500 122500]] | |||
==About this Structure== | ==About this Structure== | ||
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==Reference== | ==Reference== | ||
Klotho-related protein is a novel cytosolic neutral beta -glycosylceramidase., Hayashi Y, Okino N, Kakuta Y, | Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase., Hayashi Y, Okino N, Kakuta Y, Shikanai T, Tani M, Narimatsu H, Ito M, J Biol Chem. 2007 Oct 19;282(42):30889-900. Epub 2007 Jun 26. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17595169 17595169] | ||
[[Category: Beta-glucosidase]] | [[Category: Beta-glucosidase]] | ||
[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
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[[Category: novel cytosolic neutral beta-glycosylceramidase]] | [[Category: novel cytosolic neutral beta-glycosylceramidase]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:07:40 2008'' | ||
Revision as of 18:07, 21 February 2008
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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-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 beta-glucosidase-like domain but whose natural substrates are 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 small interfering RNA 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 k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. 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 fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.
DiseaseDisease
Known disease associated with this structure: Corticosteroid-binding globulin deficiency OMIM:[122500]
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, Shikanai T, Tani M, Narimatsu H, Ito M, J Biol Chem. 2007 Oct 19;282(42):30889-900. Epub 2007 Jun 26. PMID:17595169
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