Crystal structure of Medicago truncatula UGT85H2--insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase.

(Iso)flavonoids are a diverse group of plant secondary metabolites with important effects on plant, animal and human health. They exist in various glycosidic forms. Glycosylation, which may determine their bioactivities and functions, is controlled by specific plant uridine diphosphate glycosyltransferases (UGTs). We describe a new multifunctional (iso)flavonoid glycosyltransferase, UGT85H2, from the model legume Medicago truncatula with activity towards a number of phenylpropanoid-derived natural products including the flavonol kaempferol, the isoflavone biochanin A, and the chalcone isoliquiritigenin. The crystal structure of UGT85H2 has been determined at 2.1 A resolution, and reveals distinct structural features that are different from those of other UGTs and related to the enzyme's functions and substrate specificities. Structural and comparative analyses revealed the putative binding sites for the donor and acceptor substrates that are located in a large cleft formed between the two domains of the enzyme, and indicated that Trp360 may undergo a conformational change after sugar donor binding to the enzyme. UGT85H2 has higher specificity for flavonol than for isoflavone. Further substrate docking combined with enzyme activity assay and kinetic analysis provided structural insights into this substrate specificity and preference.

A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula.

Analysis of over 200,000 expressed sequence tags from a range of Medicago truncatula cDNA libraries resulted in the identification of over 150 different family 1 glycosyltransferase (UGT) genes. Of these, 63 were represented by full length clones in an EST library collection. Among these, 19 gave soluble proteins when expressed in E. coli, and these were screened for catalytic activity against a range of flavonoid and isoflavonoid substrates using a high-throughput HPLC assay method. Eight UGTs were identified with activity against isoflavones, flavones, flavonols or anthocyanidins, and several showed high catalytic specificity for more than one class of (iso)flavonoid substrate. All tested UGTs preferred UDP-glucose as sugar donor. Phylogenetic analysis indicated that the Medicago (iso)flavonoid glycosyltransferase gene sequences fell into a number of different clades, and several clustered with UGTs annotated as glycosylating non-flavonoid substrates. Quantitative RT-PCR and DNA microarray analysis revealed unique transcript expression patterns for each of the eight UGTs in Medicago organs and cell suspension cultures, and comparison of these patterns with known phytochemical profiles suggested in vivo functions for several of the enzymes.

Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula.

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDP-glucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant, animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered bioactive compounds.

Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula.

The biosynthesis of triterpene saponins is poorly characterized in spite of the importance of these glycosylated secondary metabolites for plant defense and animal health. The model legume Medicago truncatula synthesizes more than 30 different saponins based on at least five triterpene aglycones; soyasapogenols B and E, medicagenic acid, hederagenin and bayogenin. We have employed an inducible cell culture system, DNA array-based and in silico transcript profiling, and targeted metabolite profiling, to identify triterpene glycosyltransferases (GTs) from among the more than 300 GTs expressed in M. truncatula. Two uridine diphosphate glucosyltransferases were functionally characterized; UGT73K1 with specificity for hederagenin and soyasapogenols B and E, and UGT71G1 with specificity for medicagenic acid. The latter enzyme also glycosylated certain isoflavones and the flavonol quercetin with higher efficiency than triterpenes; however, integrated transcript and metabolite profiling supported a function for UGT71G1 in terpenoid but not (iso)flavonoid biosynthesis in the elicited cell cultures.

Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis.

Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope- and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.

A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula.

The saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin. These aglycones are most likely derived from beta-amyrin, a product of the cyclization of 2,3-oxidosqualene. Mining M. truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and beta-amyrin synthase (beta-AS). SS was functionally characterized by expression in Escherichia coli, two forms of SE by complementation of the yeast erg1 mutant, and beta-AS by expression in yeast. Beta-amyrin was the sole product of the cyclization of squalene epoxide by the recombinant M. truncatulabeta-AS, as judged by GC-MS and NMR. Transcripts encoding beta-AS, SS and one form of SE were strongly and co-ordinately induced, associated with accumulation of triterpenes, upon exposure of M. truncatula cell suspension cultures to methyl jasmonate. Sterol composition remained unaffected by jasmonate treatment. Molecular verification of induction of the triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA array-based approaches.

The phenylpropanoid pathway and plant defence-a genomics perspective.

Summary The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex flavonoids, isoflavonoids, and stilbenes. The enzymatic steps involved in the biosynthesis of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned. Less is understood about the regulatory genes that orchestrate rapid, coordinated induction of phenylpropanoid defences in response to microbial attack. Many of the biosynthetic pathway enzymes are encoded by gene families, but the specific functions of individual family members remain to be determined. The availability of the complete genome sequence of Arabidopsis thaliana, and the extensive expressed sequence tag (EST) resources in other species, such as rice, soybean, barrel medic, and tomato, allow, for the first time, a full appreciation of the comparative genetic complexity of the phenylpropanoid pathway across species. In addition, gene expression array analysis and metabolic profiling approaches make possible comparative parallel analyses of global changes at the genome and metabolome levels, facilitating an understanding of the relationships between changes in specific transcripts and subsequent alterations in metabolism in response to infection.