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

Abstract

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...

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

“…A high resolution crystal structure of murine GALC has recently been reported. [3] Soaking of crystalized enzyme with ABP 3 should yield an explanation for the noted differences in affinity of inhibitor 1 and ABPs 2-4.…”

“…[3] We examined the pH-dependence of GALC labeling by Bodipyfunctionalized ABP 3 by exposing the recombinant enzyme to the probe in buffers of varying pH ( Figure 2E). The intensity of fluorescent labeling of GALC is highest at pH 4-5 while it is almost completely abolished at pH 3 and lower or pH 7 and higher.…”

“…[2] Crystal studies indicate that no dissociation of these subunits occurs. [3] Substrate hydrolysis by GALC occurs through a Koshland double displacement mechanism with overall retention of the β-anomeric configuration of the released galactopyranoside ( Figure 1A). The two carboxylic acid residues in the active site that function as a nucleophile and a general acid/base have been identified as the glutamic acid residues E258 and E182, respectively.…”

Biochemical, cell biological, pathological, and therapeutic aspects of Krabbe's disease

“…This increased cellular uptake correlated with increased lysosomal accumulation of Tat‐GALC fusion protein in lysosomes as well as in other subcellular compartments (Zhang et al, ; Meng et al, ). Because GALC has optimal enzyme activity at lysosomal low pH (Hill et al, ), additional or alternative approaches for specific targeting or enrichment of GALC to lysosomes are required for successful ERT in KD.…”

Chaperones as potential therapeutics for Krabbe disease

“…Specifically, Hill and coworkers () identified some chemical characteristics that, associated with azasugar compound, are required for PC specificity and pH‐dependent affinity for GALC. This work was made possible by recent studies on not only the structures of GALC but also the mode of binding of substrate, intermediate, and product to the enzyme active site, accompanied by conformational changes of active site residues (Deane et al, ; Hill et al, ). Among the iminosugar, 4‐epi‐isofagomine has been reported by Biela‐Banas et al () as a suitable lead compound in the search for potential PCs because this molecule was not selective for GALC; an elaboration of this structure must be made.…”