2e9m
From Proteopedia
|
Crystal Structure of human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Prote:KLrP) complex with Galactose and fatty acids
Contents |
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.
Disease
Known disease associated with this structure: Transcortin deficiency OMIM:[122500]
About this Structure
2E9M is a Single protein structure of sequence from Homo sapiens with GAL, PLM and OLA as ligands. Active as Beta-glucosidase, with EC number 3.2.1.21 Full crystallographic information is available from OCA.
Reference
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 Mon Nov 12 21:47:16 2007
