Thioglycoligases: Mutant Glycosidases for Thioglycoside Synthesis

Jahn, Michael; Marles, Jennifer; Warren, R. A. J.; Withers, Stephen G.

doi:10.1002/anie.200390114

Cited by 150 publications

(93 citation statements)

References 28 publications

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“…Such a mechanism in which the ''base'' residue is tailored to match the pK a of the acceptor is not without precedent. Studies with mutant glycosidases have shown that removal of the charged base engineers acceptance of nonnatural sulfur-containing acceptors in transglycosylation reactions (22). It may also be relevant that the two characterized cytokinin-N-glycosyltransferases in Arabidopsis (UGT76C2 and UGT76C1) are rare examples of UGTs that do not have, based on sequence alignments, a histidine at the ''base'' position.…”

Section: Discussionmentioning

confidence: 99%

Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

Brazier-Hicks

Offen

Gershater

et al. 2007

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

275

294

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The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable Nglucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 Å, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.enzymology ͉ glycosyltransferase ͉ xenobiotic ͉ glycosides ͉ domain-swapping P lants are constantly exposed to synthetic compounds, such as pollutants and crop protection agents, and are able to transform these xenobiotics by using a four-phase detoxification system that has immediate parallels with drug metabolism in animals (Fig. 1A). Absorbed xenobiotics are first metabolically activated by ''phase 1'' enzymes, which then facilitates their subsequent bioconjugation with polar natural products (amino acids, sugars, peptides) in phase 2 metabolism. In crops and weeds, the most commonly observed phase 2 reaction is glycosylation (1), a reaction catalyzed by family GT1 glycosyltransferases (2), which are more normally engaged in secondary metabolism (3). A diverse range of xenobiotics are known to undergo conjugation as O-, S-, and N-acceptors, with UDP-glucose (UDP-glc) being the most commonly observed sugar donor (1). Once synthesized, conjugates accumulate transiently in the cytosol before being transported (phase 3) to either the vacuole or apoplast (Fig. 1 A).Despite their central importance in the metabolism of herbicides, pesticides, and organic pollutants, the identity of the enzymes catalyzing the glycosylation of xenobiotics has only recently been determined through studying their activity in the model plant Arabidopsis thaliana (4,5). Arabidopsis plants rapidly metabolize persistent pollutants such as 2,4,5-trichlorophenol (TCP) and 3,4-dichloroaniline (DCA) by O-and N-glucosylation, respectively (4-7) (Fig. 1B). Several UDP-glc-dependent glycosyltransferases (UGTs) in Arabidopsis have been shown to have O-gluco...

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Section: Discussionmentioning

confidence: 99%

Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

Brazier-Hicks

Offen

Gershater

et al. 2007

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

275

294

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“…tumor growth, progression and spread. Within this quest, thioglycosides attract attention as potential drug leads due to their stability against hydrolysis [10,11].…”

Section: Introductionmentioning

confidence: 99%

Conformational analysis of thioglycoside derivatives of histo-blood group ABH antigens using an ab initio-derived reparameterization of MM4: implications for design of non-hydrolysable mimetics

Strino

Lii

Gabius

et al. 2009

J Comput Aided Mol Des

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Histo-blood group ABH antigens serve as recognition sites for infectious microorganisms and tissue lectins in intercellular communication, e.g. in tumor progression. Thus, they are of interest as a starting point for drug design. In this respect, potent non-hydrolysable derivatives such as thioglycosides are of special interest. As prerequisite to enable estimations of ligand properties relative to their natural counterparts, conformational properties of the thioglycosidic derivatives of ABH trisaccharides and their disaccharide units were calculated using systematic and filtered systematic searches with the MM4 force field. Parameters for the glycosidic torsions of thioglycosides were independently derived from ab initio calculations. The resulting energy deviations required a reparameterization of MM4 to a new parameter set called MM4R. The data sets obtained using MM4R reveal that the thioglycosides have somewhat increased levels of flexibility about the major low-energy conformations shared with the corresponding O-glycosides. In the trisaccharides, the thiosubstitution of the Gal[NAc]alpha1-3Gal linkage leads to a preference for a conformation which is the secondary minimum of the natural counterparts. This conformation also generates contacts between the N-acetyl group and the fucose moiety in the blood group A derivative. Calculations further indicate that thiosubstitution of only the Fucalpha1-2Gal linkage does not affect the conformational preferences compared to the natural trisaccharide. Thiosubstitution of both linkages in the trisaccharide results in increased flexibility but the favored conformation of the natural trisaccharides is preferred. The study suggests that thioglycoside derivatives of ABH antigens could have pharmaceutical interest as ligands of lectins and other carbohydrate-binding proteins.

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“…These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial uses in biobleaching of paper pulp (2,3), bioconversion of lignocellulose material to fermentative products (4), improvement of animal feedstock digestibility, and recently in the field of oligosaccharide and thioglycoside synthesis (5,6).…”

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confidence: 99%

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Bravman

Belakhov

Solomon

et al. 2003

Journal of Biological Chemistry

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␤-D-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a ␤-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ϳ10 6 -fold, and this activity was enhanced 10 3 -fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu 335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a ). The mutant exhibited 10 3 -fold reduction in activity, and the Brønsted plot of log(k cat ) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 10 3 -fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat /K m ), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a ) but decreased 10 2 -fold for those that require strong acid catalysis (high pK a ). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp 495 acting as the acid-base in XynB2.

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Thioglycoligases: Mutant Glycosidases for Thioglycoside Synthesis

Abstract: Engineering of the active site of retaining glycosidases in conjunction with the appropriate modification of their substrates has led to the enzymatic synthesis of thioglycosidic linkages in thiooligosaccharides (see scheme).

Cited by 150 publications

References 28 publications

Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

Conformational analysis of thioglycoside derivatives of histo-blood group ABH antigens using an ab initio-derived reparameterization of MM4: implications for design of non-hydrolysable mimetics

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

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