X-ray and Biochemical Analysis of N370S Mutant Human Acid β-Glucosidase

Wei, Ronnie R.; Hughes, Heather; Boucher, Susan; Bird, Julie; Guziewicz, Nicholas; Patten, Scott M. Van; Qiu, Huawei; Pan, Clark Q.; Edmunds, Tim

doi:10.1074/jbc.m110.150433

Cited by 58 publications

(82 citation statements)

References 34 publications

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“…S4A) but do not require significant conformational change of active site loops, indicating that the substrate binding pocket of GALC is highly preorganized. This is in contrast to the considerable conformational changes that occur in loops around the active site of the related enzyme β-glucocerebrosidase (25,37). The scissile bond and 4-nitrophenyl aglycone project from the surface of the enzyme and contribute very little to substrate specificity or recognition (Fig.…”

Section: ) (33 34)mentioning

confidence: 55%

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Hill

Graham

Read

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme β-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of highresolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzymesubstrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.β-galactosylceramidase | lysosomal storage disease | glycosyl hydrolase | pharmacological chaperone therapy T he recycling and degradation of eukaryotic membrane components occurs in the lysosome and is essential for cellular maintenance. The molecular mechanisms of lysosomal lipid degradation are primarily informed by the study of a class of human diseases, sphingolipidoses, which are caused by inherited defects in glycosphingolipid catabolism. Krabbe disease is a devastating neurodegenerative disorder that is caused by deficiencies in the lysosomal enzyme β-galactocerebrosidase (GALC) (enzyme commission 3.2.1.46). It is essential for the catabolism of galactosphingolipids, including the principal lipid component of myelin, β-D-galactocerebroside ( Fig. 1A) (1). GALC function has also been implicated in cancer cell metabolism, primary open-angle glaucoma and the maintenance of a hematopoietic stem cell niche (2-4).GALC catalyzes the hydrolysis of β-D-galactocerebroside to β-D-galactose and ceramide, as well as the breakdown of psychosine to β-D-galactose and sphingosine. In both cases, removal of the galactosyl moiety is thought to occur via a retaining twostep glycosidic bond hydrolysis reaction (5, 6). Our recent structure of murine GALC identified two active site glutamate residues geometrically consistent with this mechanism (7). In the first step, the carboxylate group of E258 is hypothesized to perform a nucleophilic attack at ring position C 1 , forming an enzymesubstrate intermediate, releasing the first product (ceramide or sphingosine) as the leaving group. In the second step, E182 is thought to act as a general acid/base to deprotonate a water molecule, which then attacks the ring, releasing the enzyme and the second product (galactose).Defects in GALC lead to the accumulation of cytotoxic metabolites that elicit complex, and still only partially under...

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Section: ) (33 34)mentioning

confidence: 55%

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Hill

Graham

Read

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…1B). These data show that the major effects of IFG are to stabilize the mutant GCase protein in fibroblasts, suggesting that relatively minor conformational changes affect the catalytic rate constant as evidenced by the N370S structure (24). For the highly unstable L444P enzyme, IFG treatment did increase the amount of GCase protein but had little to no effect on the catalytic properties of the enzyme.…”

Section: Ifg Inhibition Of Gcases-mentioning

confidence: 77%

Ex Vivo and in Vivo Effects of Isofagomine on Acid β-Glucosidase Variants and Substrate Levels in Gaucher Disease

Sun

Liou

et al. 2012

Journal of Biological Chemistry

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Background: "Chaperones" may enhance mutant enzyme activities, but therapeutic levels have not been shown in vivo. Results: A chaperone, isofagomine, stabilizes wild type and mutant acid ␤-glucosidases in tissues and sera and reduces visceral substrates in vivo. Conclusion: These effects are enhanced pre-versus postsynthetically. Significance: The results are proof of principle for the potential therapeutic use in residual enzyme diseases.

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“…Analysis of the loops surrounding the active site suggests that GALC is unlikely to undergo a conformational change similar to that described for GlcCerase in order to accommodate substrate (Fig. S6) (18,19). The scissile bond points out toward the surface of the protein but in order to gain access to the membrane-embedded substrate, glycolipid hydrolases require saposins (saps) (20).…”

Section: Discussionmentioning

confidence: 99%

Insights into Krabbe disease from structures of galactocerebrosidase

Deane

Graham

Kim

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

118

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Krabbe disease is a devastating neurodegenerative disease characterized by widespread demyelination that is caused by defects in the enzyme galactocerebrosidase (GALC). Disease-causing mutations have been identified throughout the GALC gene. However, a molecular understanding of the effect of these mutations has been hampered by the lack of structural data for this enzyme. Here we present the crystal structures of GALC and the GALC-product complex, revealing a novel domain architecture with a previously uncharacterized lectin domain not observed in other hydrolases. All three domains of GALC contribute residues to the substrate-binding pocket, and disease-causing mutations are widely distributed throughout the protein. Our structures provide an essential insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation for the severity of many mutations associated with fatal infantile disease. The localization of disease-associated mutations in the structure of GALC will facilitate identification of those patients that would be responsive to pharmacological chaperone therapies. Furthermore, our structure provides the atomic framework for the design of such drugs.

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X-ray and Biochemical Analysis of N370S Mutant Human Acid β-Glucosidase

Cited by 58 publications

References 34 publications

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

Ex Vivo and in Vivo Effects of Isofagomine on Acid β-Glucosidase Variants and Substrate Levels in Gaucher Disease

Insights into Krabbe disease from structures of galactocerebrosidase

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