Plant polysaccharides
represent a virtually unlimited feedstock
for the generation of biofuels and other commodities. However, the
extraordinary recalcitrance of plant polysaccharides toward breakdown
necessitates a continued search for enzymes that degrade these materials
efficiently under defined conditions. Activity-based protein profiling
provides a route for the functional discovery of such enzymes in complex
mixtures and under industrially relevant conditions. Here, we show
the detection and identification of β-xylosidases and
endo
-β-1,4-xylanases in the secretomes of
Aspergillus niger
, by the use of chemical probes inspired
by the β-glucosidase inhibitor cyclophellitol. Furthermore,
we demonstrate the use of these activity-based probes (ABPs) to assess
enzyme–substrate specificities, thermal stabilities, and other
biotechnologically relevant parameters. Our experiments highlight
the utility of ABPs as promising tools for the discovery of relevant
enzymes useful for biomass breakdown.
Gaucher
disease is caused by inherited deficiency in glucocerebrosidase
(GBA, a retaining β-glucosidase), and deficiency in GBA constitutes
the largest known genetic risk factor for Parkinson’s disease.
In the past, animal models of Gaucher disease have been generated
by treatment with the mechanism-based GBA inhibitors, conduritol B
epoxide (CBE), and cyclophellitol. Both compounds, however, also target
other retaining glycosidases, rendering generation and interpretation
of such chemical knockout models complicated. Here we demonstrate
that cyclophellitol derivatives carrying a bulky hydrophobic substituent
at C8 are potent and selective GBA inhibitors and that an unambiguous
Gaucher animal model can be readily generated by treatment of zebrafish
with these.
α-d-Gal-cyclophellitol cyclosulfamidate is a new class of neutral, conformationally-constrained competitive glycosidase inhibitor that stabilizes α-gal A and prevents its degradation both in vitro and in cellulo by mimicry of the Michaelis complex conformation.
The lysosomal glycoside hydrolase -glucocerebrosidase (GBA; sometimes called GBA1 or GC ase ) catalyses the hydrolysis of glycosphingolipids. Inherited deficiencies in GBA cause the lysosomal storage disorder Gaucher disease (GD). Consequently, GBA is of considerable medical interest, with continuous advances in the development of inhibitors, chaperones and activity-based probes. The development of new GBA inhibitors requires a source of active protein; however, the majority of structural and mechanistic studies of GBA today rely on clinical enzyme-replacement therapy (ERT) formulations, which are incredibly costly and are often difficult to obtain in adequate supply. Here, the production of active crystallizable GBA in insect cells using a baculovirus expression system is reported, providing a nonclinical source of recombinant GBA with comparable activity and biophysical properties to ERT preparations. Furthermore, a novel crystal form of GBA is described which diffracts to give a 0.98 Å resolution unliganded structure. A structure in complex with the inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro--d-glucopyranoside was also obtained, demonstrating the ability of this GBA formulation to be used in ligand-binding studies. In light of its purity, stability and activity, the GBA production protocol described here should circumvent the need for ERT formulations for structural and biochemical studies and serve to support GD research.
Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activitybased probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N-functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6-and N-functionalised reagents, a bi-functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.