Article16 February 2006free access Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification Wendy Offen Wendy Offen York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Carlos Martinez-Fleites Carlos Martinez-Fleites York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Min Yang Min Yang Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Eng Kiat-Lim Eng Kiat-Lim CNAP, Department of Biology, University of York, York, UK Search for more papers by this author Benjamin G Davis Benjamin G Davis Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Chris A Tarling Chris A Tarling Department of Chemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Christopher M Ford Christopher M Ford School of Agriculture, Food and Wine, The University of Adelaide, Australia Search for more papers by this author Dianna J Bowles Dianna J Bowles CNAP, Department of Biology, University of York, York, UK Search for more papers by this author Gideon J Davies Corresponding Author Gideon J Davies York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Wendy Offen Wendy Offen York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Carlos Martinez-Fleites Carlos Martinez-Fleites York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Min Yang Min Yang Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Eng Kiat-Lim Eng Kiat-Lim CNAP, Department of Biology, University of York, York, UK Search for more papers by this author Benjamin G Davis Benjamin G Davis Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Chris A Tarling Chris A Tarling Department of Chemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Christopher M Ford Christopher M Ford School of Agriculture, Food and Wine, The University of Adelaide, Australia Search for more papers by this author Dianna J Bowles Dianna J Bowles CNAP, Department of Biology, University of York, York, UK Search for more papers by this author Gideon J Davies Corresponding Author Gideon J Davies York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK Search for more papers by this author Author Information Wendy Offen1, Carlos Martinez-Fleites1, Min Yang2, Eng Kiat-Lim3, Benjamin G Davis2, Chris A Tarling4, Christopher M Ford5, Dianna J Bowles3 and Gideon J Davies 1 1York Structural Biology Laboratory, Department of Chemistry, University of York, York, UK 2Department of Chemistry, University of Oxford, Oxford, UK 3CNAP, Department of Biology, University of York, York, UK 4Department of Chemistry, University of British Columbia, Vancouver, BC, Canada 5School of Agriculture, Food and Wine, The University of Adelaide, Australia *Corresponding author. York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK. Tel.: +44 1904 328260; Fax: +44 1904 328266; E-mail: [email protected] The EMBO Journal (2006)25:1396-1405https://doi.org/10.1038/sj.emboj.7600970 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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-O-glycosyltransferase (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. Introduction Plants have evolved an extraordinary capacity to perceive changes in their environment and respond rapidly to maximize opportunity and minimize risk. The plasticity of these responses requires the integration of growth, development and metabolism, which in turn has led to the evolution of diverse mechanisms to regulate cellular homeostasis. Glycosylation is one of these mechanisms, with a large multigene family of glycosyltransferases (GTs) able to recognize lipophilic small molecules including hormones, secondary metabolites and both biotic and abiotic toxins in the environment (Lim and Bowles, 2004; Bowles et al, 2005). The plant enzymes belong to the ‘GT1 family’ in the CAZy classification of carbohydrate-active enzymes (Couthino et al, 2003), which also includes the mammalian glucuronosyltransferases, similarly involved in cellular homeostasis and the metabolism of pharmaceuticals and dietary compounds of clinical and nutritional relevance (Dutton, 1980; Tukey and Strassburg, 2000). There is a substantial set of gene sequence data for GT1 enzymes with over 1200 open reading frames currently defined (December 2005). In the case of plants, the phylogenetics of the large multigene family in Arabidopsis thaliana has been analysed and compared to the evolution of substrate recognition (Lim et al, 2003). Studies have revealed that the plant GTs are highly regio- and stereo-selective, but are often capable in vitro of recognizing common features on multiple substrates, whether hormones, secondary metabolites or xenobiotics, a promiscuity which is of direct physiological significance for the plasticity of response (Lim and Bowles, 2004; Bowles et al, 2005). While regio- and stereo-selectivity can be defined using different naturally occurring aglycones and their analogues, it has not been possible to understand the molecular basis of donor and acceptor specificity for these reactions. Structures of several bacterial GTs of the GT1 family involved in vancomycin synthesis have been solved and shown to have the twin Rossmann-domain ‘GT-B’ fold (Mulichak et al, 2001, 2003, 2004), but the sequences, of these enzymes of known structure, have less than 5% identity to the plant and mammalian enzymes of the same family. One of the most significant, and representative, glycosylation reactions in plants is the formation of anthocyanins. Anthocyanins are water-soluble pigments based on a tricyclic flavonoid core. In the plant, the compounds function as colourants determining flower and fruit colour. During red winemaking, anthocyanins are progressively extracted into the aqueous must, which reaches maximum colour intensity at around 6 days. The colour thereafter decreases due to a loss of copigmentation (intramolecular H-bond mediated stacking of anthocyanins with nonpigmented flavonoid or phenolic partners, interrupted by increasing alcoholic concentration as fermentation proceeds) and the formation of pigmented polymeric compounds through chemical reactions with proanthocyanidin (tannin) monomers progressively extracted from skins and seeds as the percentage of alcohol increases. After 6–12 months, almost no free anthocyanins remain in wine and all colour is derived from the presence of pigmented polymeric anthocyanin:tannin compounds (Fulcrand et al, 2004). Increasingly, flavonoids are recognized for their health-related properties, both in the human diet and for the improvement of animal feed. A number of clinical effects have been ascribed to different plant flavonoids, including antitumour, anti-inflammatory and antimicrobial properties (Ross and Kasum, 2002; Halliwell et al, 2005). In the red grape (Vitis vinifera L., cv. Shiraz), cyanidin, an unstable precursor, is glycosylated by a uridine diphospho (UDP)-glucose:flavonoid 3-O-glycosyltransferase (VvGT1) to yield the stable anthocyanin, cyanidin 3-O-glucoside (Figure 1). Glucosylation leads to transport of the product from the cytosol into the vacuole where levels accumulate as the fruit ripens. VvGT1 is also active on a number of different flavonoids in vitro including quercetin and kaempferol, with rates in vitro ca. 2 and 0.6% of the cyanidin acceptor. The sequence of red grape GT has ca 60% identity with the 78D clade of A. thaliana GTs, and >50% identity to related enzymes in other species including those of petunia and strawberry, and thus serves as a powerful template upon which to illuminate substrate specificity across the whole family. Figure 1.(A) The reaction catalysed by Vitis vinifera UDP-Glucose:flavonoid 3-O-glycosyltransferase is the addition of glucose, from a UDP-glucose donor to the 3-O position of cyanidin. The reaction occurs with inversion of anomeric configuration to generate the β-glucoside. (B) Vitis vinifera cyanidin 3-O-glycosyltransferase will also accommodate quercetin and kaempferol as acceptors at 2 and 0.6% of the rate with the natural acceptor (Ford et al, 1998). The numbering of the oxygens, as used in the text, is shown for kaempferol. Download figure Download PowerPoint Here we present the 3-D structure, interpreted in light of ligand complexes, ‘high-throughput’ screening of catalytic activity, and kinetics of wild type and mutant enzymes of the plant GT1 involved in the glucosylation of cyanidins in red grapes. The 3-D structure of VvGT1 has been solved at 1.9 Å resolution in a UDP (product) bound form and, subsequently, in its ‘Michaelis’ complex with both an intact UDP-Glc donor and the acceptor kaempferol, also at 1.9 Å resolution and in ‘nonproductive’ complex with UDP and quercetin, at 2.1 Å. These three-dimensional structures lay the foundation for detailed dissection of catalytic mechanism and specificity, and should, in harness with recent structural work elsewhere (Shao et al, 2005), inform subsequent exploitation of the many plant GT1 enzymes. Results Screening for VvGT1 activities in vitro The gene encoding VvGT1 was expressed as a recombinant protein in Escherichia coli and purified to electrophoretic homogeneity. In vivo, VvGT1 functions as a flavonoid 3-O-glycosyltransferase during the formation of red wine pigments. The Green-Amber-Red ‘GAR’ screen (Yang et al, 2005a) was used to probe alternative substrates, in vitro, using a large acceptor and nucleotide-sugar donor screen. Liquid Chromatography Mass Spectrometry (LCMS), with an internal guanosine diphosphate (GDP) standard, was thus used to screen a large and representative donor and acceptor library in multi-well format trays. A virtual colour, reflecting DNA micro-array practice, of red (no reaction), amber (total ion-count (TIC) signal/noise >1) and green (TIC signal-to-noise >10) is assigned reflecting the presence of selected ions of mass n+y where y is the mass of the glycosyl moiety transferred (Flint et al, 2005; Yang et al, 2005a). Full, ‘pseudo-single substrate’ kinetics, chosen to probe both nucleotide and sugar specificities, were subsequently performed on the panel of viable different sugar donors identified in the high-throughput screen. VvGT1 native enzyme kinetics Under the conditions of the mass-spectrometry based kinetic assay, VvGT1 transfers Glc from UDPGlc to Quercetin and kaempferol at around 0.08 s−1 with a Km for UDP-Glc of 680 μM and for quercetin and kaempferol of 31 and 42 μM (Table I). In addition to the favoured donor, UDP-Glc, VvGT1 can also harness a wide panel of different UDP-sugars: UDP-5SGlc, UDP-Xyl, UDP-Man, UDP-Gal and UDP-GlcNAc (Table I), as well as GDP-Glc and dTDP-Xyl. VvGT1 showed no activity with UDP-6OMeGal, UDP-Ara, UDP-6FGal, UDP-GlcN, UDP-2FGal, UDP-5SAra, GDP-Man and neither GDP-Fuc nor UDP-Fuc. Importantly, VvGT1 does not process the sugar donor UDP-Rha, which is highly important in plant secondary metabolism. The transfer rates for these different sugar donors reflects changes both in kcat and Km (Table I). Simply in terms of kcat, UDP is clearly the favoured nucleotide, with dTDP forms of both glucose and xylose transferred approximately five times more slowly than their UDP counterparts. GDP-Glc is accepted at approximately 1/100th the rate of UDP-Glc. The 6-hydroxyl of the donor is of lesser importance for catalysis, with UDP-Xyl transferred sixty times more slowly than UDP-Glc while, in contrast, there is a distinct epimeric preference at O4: UDP-Gal is transferred 210 times more slowly (in terms of kcat) than UDP-Glc with kcat/Km reduced some 16-fold. Table 1. Kinetic data of VvGT1 and mutants Enzyme Substrate varied Substrate fixed Km/μM kcat (s−1) kcat/Km (s−1 μM−1) WT UDPGlc Quercetin 679±74 8.4 × 10−2±9.1 × 10−3 124±13 WT dTDPGlc Quercetin 300±35 1.6 × 10−2±1.8 × 10−3 52±6.0 WT UDP5SGlc Quercetin 166±1.7 8.8 × 10−4±9.0 × 10−6 5.3±0.05 WT UDPGlcNAc Quercetin 194±23.2 1.04 × 10−3±1.2 × 10−4 5.3±0.6 WT UDPGal Quercetin 48.2±0.5 3.8 × 10−4±3.9 × 10−6 7.9±0.08 WT UDPMan Quercetin 50.1±5.0 1.0 × 10−5±1.0 × 10−6 0.2±0.02 WT GDPGlc Quercetin 167±2.60 9.7 × 10−4±1.5 × 10−5 5.8±0.09 WT dTDPXyl Quercetin 166±8.0 2.5 × 10−4±0.12 × 10−5 1.5±0.07 WT UDPXyl Quercetin 219±22 1.4 × 10−3±1.4 × 10−4 6.2±0.6 WT Quercetin UDPGlc 30.8±1.5 7.5 × 10−2±3.7 × 10−3 2437±118 WT Kaempferol UDPGlc 42.3±2.1 1.7 × 10−2±8.9 × 10−3 391±19 Q375N UDPGlc Quercetin 529±55 7.6 × 10−4±9.0 × 10−5 1.6±0.2 Q375H UDPGlc Quercetin 253±30 1.0 × 10−4±1.1 × 10−5 0.4±0.05 T141A UDPGlc Quercetin 310±28 1.5 × 10−2±1.3 × 10−3 49±4.5 T141A UDPGal Quercetin 120±15 8.1 × 10−4±1.0 × 10−4 6.7±0.83 Q375N/T141A UDPGlc Quercetin 120±10 7.0 × 10−4±5.8 × 10−5 5.8±0.49 Q375N/T141A UDPGal Quercetin 33±2.0 6.5 × 10−5±3.9 × 10−6 1.9±0.11 H20A UDPGlc/Gal/Rha Quercetin N/D D374A UDPGlc/Gal/Rha Quercetin N/D N/D, no detectable activity. A large panel of acceptors (as described fully in Flint et al, 2005; Yang et al, 2005a) was subsequently used to probe the acceptor tolerance of VvGT1 using the favored UDP-Glc as donor. While, in vivo, VvGT1 is a 3-O-glycosyltransferase, it was able to transfer to a spectrum of different compounds (Figure 2), including some whose sole hydroxyl is equivalent to the O7 of the natural acceptors. This strongly suggests that, in vitro, VvGT1 can be persuaded to accept a wide variety of flavonoid and similar acceptors; it is unclear how these preferences would manifest themselves in vivo, where cellular location and the availability and concentration of donors and acceptors would be major factors for catalysis. The relative catalytic promiscuity of VvGT1 adds to the emerging picture (Davies et al, 2005; Flint et al, 2005) that many GTs are less specific, and thus also amenable to exploitation in biocatalysis, than might have, at one time, been assumed (since much of the pioneering work in the glycosyltransferase field was performed on enzymes which show much stricter specificity, exemplified by those enzymes involved in blood group synthesis (Patenaude et al, 2002; Marcus et al, 2003)). Figure 2.An example of the Green-Amber-Red screen for acceptor specificity using UDP-Glc as the donor. (A) A virtual microarray with red indicating no reaction, amber total ion-count signal/noise >1 and green signal-to-noise >10. (B) The panel of different acceptors identified with TIC signal-to-noise >10. A similar approach led to the identification of a spectrum of potential nucleotide sugar donors (see text). Download figure Download PowerPoint Structure determination of the UDP-bound form of VvGT1 The structure of VvGT1 was solved initially using 1.9 Å data in complex with UDP in tandem with 2.2 Å selenomethionine data, the latter collected so as to optimize the f″ signal from the Se anomalous scatterers (see Materials and methods). Subsequently, crystals were obtained both of the ‘Michaelis’ complex of nontransferable UDP-2-deoxy-2-fluoro glucose donor (UDP-2FGlc, synthesized according to Gibson et al, 2004) with the acceptor kaempferol (Figure 1B) and the ‘abortive’ complex of UDP with quercetin. The structure of UDP: VvGT1, a monomer in both crystal and solution, may be traced from Asn7 to Val456 with two regions (His54-Met60; Pro251-Thr259) disordered in electron density. The structure forms two ‘Rossmann-like’ (β/α/β) domains, comprising residues 7–250 and 260–437, respectively (Figure 3A) and termed the ‘GT-B’ fold. As with other GT-B structures (reviewed in Hu and Walker, 2002), the final C-terminal helix crosses from the C-terminal domain to complete the N-terminal domain fold through residues 437–456. This fold is similar to the GT fold class ‘GT-B’ described originally for the T4 DNA β-glucosyltransferase (Vrielink et al, 1994) and subsequently in, for example, the E. coli MurG enzyme involved in peptidoglycan formation (Hu et al, 2003) and the bacterial enzymes involved in vancomycin synthesis (Mulichak et al, 2001, 2003, 2004). Figure 3.(A) Stereo (divergent) view of the Vitis vinifera UDP-Glucose:flavonoid 3-O-glycosyltransferase in Michaelis complex with UDP-2-deoxy-2-fluoro glucose (donor) and kaempferol (acceptor). The molecule is colour-ramped from N-terminus (blue) to C-terminus (red) with the ligands shown in ‘ball-and-stick’ representation. (B) Stereo (divergent) view of the overlap of two GT1 enzymes VvGT1 (blue), and the nucleotide complex of the GT1 enzyme GtfA (yellow). The region defining the C-terminal ‘signature’ motif, displayed by some GT1 enzymes, is shown in cyan for VvGt1 only. This figure was generated with MOLSCRIPT (Kraulis, 1991). Download figure Download PowerPoint To date, glycosyltransferase family GT1 contains over 1200 open reading frames. The sequences of the bacterial GT1 enzymes of known 3-D structure differ substantially from those of the plant enzymes (<5% identity). This has led some authors to consider that family GT1 contains different ‘subfamilies’ reflecting, in part, the absence of the C-terminal 44-amino-acid signature motif that is found in the vast majority of plant and mammalian GT1 enzymes (Lim and Bowles, 2004). The 3-D structure of VvGT1 shows that this signature defines the environment of the nucleotide-sugar binding site of the plant (and by implication other higher eukaryotic) enzymes. The ‘consensus’ (Lim and Bowles, 2004) sequence, is described by the following residues (with those of VvGT1 highlighted in bold): [FW332]-2X-Q-2X-[LIVMYA]-[LIMV]-46X-[LVGAC]-[LVFYA]-[LIVMF]-[STAGCM]-[HNQ]-[STAGC]-G-2X-[STAG]-3X-[STAGL]-[LIVMFA]-4X-[PQR]-[LIVMT]-3X-[PA]-3X-[DES]-[Q375EHN]. Many of these residues are simply involved in C-terminal fold ‘maintenance’, although some play roles in substrate-binding and catalysis (and thus are also present in the bacterial enzymes, below). It is, indeed, the fine details of the nucleotide pocket environment that differ between the plant enzyme (and by implication its close sequence homologs) and the Gtf series of bacterial GT1 enzymes involved in vancomycin synthesis. In particular, the conformation and composition of the loop between the 6th (final) β-strand of the C-terminal domain and its subsequent helix, residues 332–337 in VvGT1, is markedly different in the three families. This loop region contributes Trp332 to form the hydrophobic platform upon which the uracil base stacks in VvGT1. Subsequently, Gln335, while making no direct interactions with the nucleotide-sugar, is involved in maintaining Glu358 in the correct orientation to bind both ribose hydroxyls. In the bacterial enzymes, GtfA, for example, there is no stacking ‘above’ the base equivalent to Trp332 (this residue is a Glu in GtfA) nor is there an equivalent to Gln335, the comparable residue being Leu280. Further differences manifest themselves in the ribose binding region of this signature: a glutamate in this position is invariant in those GT-B fold enzymes which use UDP-sugars as donors but replaced by a hydrophobic residue (e.g. Leu301 in GtfA) in dTDP-donor enzymes reflecting the more hydrophobic nature of the deoxy-sugar (Mulichak et al, 2001, 2003, 2004). These structural changes are entirely consistent with the moderate reduction in catalytic efficiency of VvGT1 between UDP-Glc and dTDP-Glc of some 2.3 (kcat/Km) to 5 (kcat) fold (Table I). As one approaches the catalytic centre, more of the residues of the ‘signature’ motif are conserved within the wider GT1 family, including the bacterial enzymes of vancomycin synthesis. Histidine 350 interacts with O2 of the β-phosphate of UDP and Ser355 with the equivalent atom of the α-phosphate in VvGT1, the equivalents in GtfA are His293 and Thr 298. The Michaelis complex of VvGT1, below, allows us to demonstrate that in VvGT1, Asp374 and Gln375 of this signature motif are key players in sugar recognition whose sequence diversity reflects the use of different sugar donors on different enzymes. Structure of the VvGT1 Michaelis complex of UDP-2FGlc and kaempferol and its nonproductive complex with UDP and quercetin The Michaelis complex with the kaempferol acceptor and the nontransferable donor UDP-2FGlc (Figures 4A and 5) reveals the crucial interactions of the flavonoid acceptors and sugar nucleotide donors that are central to catalysis and specificity. Recognition of the O2 and O3 of glucose is conferred through the NH2 of Gln375 interacting with both O2 (F2) and O3 hydroxyls, while Asp374 hydrogen-bonds to both O3 and O4 through its OD1 and OD2 oxygens, respectively. The mutation D374A abolishes detectable catalytic activity (Table I), while the Q375N and Q375H mutations show seriously impaired or abolished catalytic activity, respectively. The O4 hydroxyl of glucose also interacts with the main-chain amide hydrogen of Trp353. The sole direct interaction of the O6 of glucose is with OG of Thr141, mutation of this residue T141A reduces the activity just six-fold, consistent with the lesser importance of the O6 in catalysis (discussed above). Figure 4.(A) Stereo (divergent) view of the observed electron density (maximum-likelihood weighted 2Fobs−Fcalc contoured at 1σ/0.4 electrons/Å3) of the Michaelis complex of the Vitis vinifera UDP-Glucose:flavonoid 3-O-glycosyltransferase with (nontransferrable) UDP-2-deoxy-2-fluoro glucose (donor) and kaempferol (acceptor). (B) Mono view of the observed electron density (as above) for quercetin bound to VvGt1 showing the single additional interaction it makes with the enzyme, a water-mediated hydrogen bond to the main-chain carbonyl of Glu189. All other interactions of this acceptor are identical to those seen with kaempferol. This figure was drawn with BOBSCRIPT (Esnouf, 1997). Download figure Download PowerPoint Figure 5.(A) Schematic diagram of active centre of the Vitis vinifera UDP-Glucose:flavonoid 3-O-glycosyltransferase showing the role of His20 as a Brønsted base for the activation of the flavonoid O3 hydroxyl for nucleophilic attack at C1 of the donor. (B) Schematic representations of UDP-α-D-glucose in 4C1 conformation and UDP-β-L rhamnose in both its favored 1C4 conformation and in 4C1 conformation. Steric factors dictate that UDP-β-L rhamnose will exist in a conformation skewed between those drawn; one which allows an axial leaving group orientation while fulfilling the stereoelectronic considerations of an incipient oxocarbenium-ion like transition state. Download figure Download PowerPoint Although many of the donor site interactions are likely to be similar in family GT1 enzymes, the sugar donor site shows distinct differences from that previously described for UDP-GlcNAc binding to the family GT28 enzyme MurG (Hu et al, 2003). While many aspects of the two structures are similar, such as the Glu–ribose interaction (above) and the main-chain amide hydrogens and Ser/Thr hydroxyls binding the phosphates (above), the direct interactions to the sugar itself are significantly different. In both structures, there is a main-chain amide interaction with O4. The interaction equivalent to that of VvGT1 Asp374 with the O3 and O4 hydroxyls is performed in MurG by a similarly located Gln (Gln288) but while the following residue, Gln375 in VvGT1, is also a glutamine in MurG, slight main-chain rearrangements means it interacts only with O3, consistent with the 2-substituent in GlcNAc. O6 interactions of the GlcNAc MurG are quite different (it is flipped through 180 degrees relative to that in VvGT1); they occur through solvent waters and O1 of the α-phosphate, compared to the direct interaction with Thr141 in VvGT1. The N-acetyl binding pocket required by MurG, is instead occupied by a phenylalanine, Phe372, in VvGT1. This residue, preceded by an a Pro-Phe motif common to both enzymes, marks a substantial main-chain difference between the two enzymes after strand β-2 of the C-terminal domain; in MurG an extended insertion at the end of this strand prior to the subsequent helix, provides the additional pocket to accommodate the N-acetyl moiety of the substrate. No such space is available in VvGT1, which consequently must undergo conformational changes to Phe372 in order to accommodate N-acetylglucosamine (which it does with around 4% of the efficiency as with UDP-Glc). The kaempferol acceptor lies in a hydrophobic, but open-ended, pocket defined by phenylalanine residues at positions 15, 121, 200 and 372 as well as by Ile87 and Val281. The structure clearly demonstrates the difference between the plant GT1 enzymes and the bacterial GTs in terms of acceptor-binding. While the attacking hydroxyl of the kaempferol acceptor lies in exactly the same location as the reacting atom of desvancosaminyl vancomycin in GtfD (Figure 6), no other atom of the flavonoid co-locates with vancomycin and no interactions are conserved. Thus, while the central core of β-strands are topologically equivalent between the VvGT1 and GtfA structures (Figure 3B), all the interactions between acceptor and enzyme are made with the loop regions that are entirely different between the two enzymes. The acceptor binding site of VvGT1 is an open-ended canyon in which the flavonoid O7 group points ‘outwards’ into solvent (Figure 7). This topography would permit accommodation of much larger substrates, including flavonol 7-O-glycosides, into the active centre. Indeed, we observe a low occupancy of a contaminating 7-O glycoside of kaempferol with the O7 substituent projecting into solvent. Mass-spectrometry (data not shown) confirms that approximately 5% of commercial kaempferol is present as its glycosyl-adduct. This is of special significance since many naturally occuring flavonoids are indeed known to exist with extended glycan decorations at this position (Graham, 1988; Harborne and Willams, 2001) and so it strongly suggests that VvGT1 could glycosylate such compounds in vivo. The open active centre presumably also contributes to the partial acceptor promiscuity defined by the GAR screen (Figure 2), for it would suggest that ‘reversed’ binding modes (in which O3 points towards solvent and O7 towards the C1 of the donor sugar) may also be permitted, although given the totally different acceptor binding modes displayed by related GT1 enzymes glycosylating at spatially-close centres (Mulichak et al, 2001, 2003, 2004), further speculation about the exact mode of binding of these different acceptors is dangerous. Figure 6.An overlap (divergent stereo) of GtfA (family GT1, in cyan), MurG (family GT28, in green) and VvGT1 (family GT1, in grey). Ligands shown are vancomycin for GtfA, UDPGlcNAc for MurG and both UDP-2FGlc and kaempferol for VvGT1. The proposed catalytic bases are presumed to be His20 of VvGT1, Asp13 of GtfA and His19 of MurG. Download figure Download PowerPoint Figure 7.Stereo (divergent) view of the protein surface in the vicinity of the substrate-binding canyon showing that, while UDP-Glc is buried, the kaempferol acceptor is only partially occluded with O7 pointing into solvent. This figure was drawn with PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net/). Download figure Download PowerPoint The bicyclic and phenol rings of kaempferol are not co-planar. Instead, the phenolic group lies at about 30° to the bicyclic moiety as would be expected in order to minimize ‘ortho-ortho’ steric interactions between the two rings. This orientation is favoured by hydrogen-bonding of the flavonol O7 to Gln84 and the phenolic hydroxyl to His150. The attacking O3 hydroxyl of the flavonoid lies 2.7 Å from His20. This residue, and its equivalents on other GT-B fold structures (Figure 6), is believed to play the role of catalytic base for the deprotonation of O3 to allow nucleophilic attack at the anomeric centre of the donor. The geometry is exquisitely poised ‘in-line’, with an O3kaempferol-C1UDP-Glc-O1UDP-Glc angle of 160°, as expected for the Michaelis complex in a single displacement mechani
