Jacqueline Wicki

Trapping covalent intermediates on beta-glycosidases.

The mechanism-based inactivation and subsequent identification of the nucleophilic residue using mass spectrometry have been successfully applied and used to identify the active-site nucleophile in numerous beta-glycosidases, as illustrated using C. fimi exoglycanase. Evidence for a covalent glycosyl-enzyme intermediate has come from X-ray crystallographic analysis of trapped complexes, the first being that of the trapped fluoroglycosyl-enzyme intermediate of Cex. The crystal structure of the trapped fluorocellobiosyl-enzyme complex for Cex has provided useful insights into catalysis and the roles of specific residues at the active site. In addition, information about the conformation of the natural sugar in the covalently bound state and the interactions at the active site was obtained using a mutant form of Cex.

Circular Permutation of Bacillus circulans Xylanase: A Kinetic and Structural Study †

The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a beta-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within beta-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors

Summary N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C6 alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.

1-Deoxy-d-galactonojirimycins with dansyl capped N-substituents as β-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines

Graphical abstract Highlights ► N-Alkylation of 1-deoxy-D-galactonojirimycin with dansylamino terminated spacer arms provides powerful inhibitors. ► These enhance mutant lysosomal -galactosidase activities in GM1 gangliosidosis cell lines. ► Based on their fluorescence, they can serve as selective intracellular organellar probes.

1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines.

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of d-glucosidases and could also be shown to distinctly improve, at sub-inhibitory concentrations, the activity of beta-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.

Potent human α-amylase inhibition by the β-defensin-like protein helianthamide

Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides.

A fluorescent probe for GM1 gangliosidosis related β-galactosidase: N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-d-galactitol

N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol, a strong competitive inhibitor of β-galactosidase, enhances residual β-galactosidase activities in fibroblasts and serves as lead en route to diagnostic compounds for tracking the fate of mutant β-gal as well as aberrant GM1 gangliosides by live cell imaging.

Atomic Resolution Analyses of the Binding of Xylobiose-Derived Deoxynojirimycin and Isofagomine to Xylanase Xyn10A

The atomic resolution structures of xylobiose-derived isofagomine and xylobiose-derived deoxynojirimycin in complex with the xylanase Xyn10A from Streptomyces lividans reveal undistorted (4)C(1) chair conformed sugars and, in the case of the deoxynojirimycin analogue, suggest unusual pK(a) changes of the enzymes catalytic machinery upon binding.