Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP-glucose:flavonoid 3-Oglycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health-promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three-dimensional structure of VvGT1 has also been determined, both in its 'Michaelis' complex with a UDP-glucose-derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

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...

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

O-GlcNAc hydrolase, OGA, removes O-linked N-acetylglucosamine (O-GlcNAc) from myriad nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments we determined the 3-D structure of human OGA, revealing an unusual helix exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors having clinical value.

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Alternatives to petroleum-based chemicals are highly sought-after for ongoing efforts to reduce the damaging effects of human activity on the environment. Copper radical oxidases from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts because they oxidize primary alcohols in a chemoselective manner without complex organic cofactors. However, despite numerous studies on canonical galactose oxidases (GalOx, EC 1.1.3.9) and engineered variants, and the recent discovery of a Colletotrichum graminicola copper radical alcohol oxidase (AlcOx, EC 1.1.3.13), the catalytic potentials of very few AA5_2 members have been characterized. Guided by the sequence similarity network and phylogenetic analyses, we targeted a distinct paralog from the fungus C. graminicola as a representative member of a large uncharacterized subgroup of AA5_2. Through recombinant production and detailed kinetic analysis, we demonstrated that this enzyme is weakly active toward carbohydrates but efficiently catalyzes the oxidation of aryl alcohols to the corresponding aldehydes. As such, this represents the initial characterization of a demonstrable aryl alcohol oxidase (AAO, EC 1.1.3.7) in AA5, an activity which is classically associated with flavin-dependent glucose-methanol-choline (GMC) oxidoreductases of Auxiliary Activity Family 3 (AA3). X-ray crystallography revealed a distinct multidomain architecture comprising an N-terminal PAN domain abutting a canonical AA5 seven-bladed propeller catalytic domain. Of direct relevance to biomass processing, the wild-type enzyme exhibits the highest activity on the primary alcohol of 5-hydroxymethylfurfural (HMF), a product of significant interest in the lignocellulosic biorefinery concept. Thus, the chemoselective oxidation of HMF to 2,5-diformylfuran (DFF) by C. graminicola aryl alcohol oxidase (CgrAAO) from AA5 provides a fundamental building block for chemistry via biotechnology.

The elucidation and prediction of how changes in a protein give altered activities and selectivities remains a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: i) obtaining diverse datasets and ii) suitable parameter frameworks that encapsulate activities in large sets. Here we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whilst sequence analysis alone fails for GT1 substrate utilization patterns, our chemical-bioinformatic model, GT-Predict, succeeds by coupling physicochemical features with isozyme recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and allowed functional annotation for uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, informing mechanism. This multifaceted approach to enzyme prediction could guide streamlined utilization (and design) of biocatalysts and discovery of other family-wide protein functions.

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