Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07 kDa and 54.39 kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4′- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.

Caffeoyl‐coenzyme A O‐methyltransferase cDNA was cloned from dark‐grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli . The translated polypeptide of 27.1‐kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O‐methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal‐affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation‐dependent O‐methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+‐ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O‐methyltransferases.

In livingstone daisy (Dorotheanthus bellidiformis ), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.

A recently discovered, S‐adenosyl‐L ‐methionine and bivalent cation‐dependent O‐methyltransferase from the ice plant, Mesembryanthemum crystallinum , is involved in the methylation of various flavonoid and phenylpropanoid conjugates. Differences in regiospecificity as well as altered kinetic properties of the recombinant as compared to the native plant O‐methyltransferase can be attributed to differences in the N‐terminal part of the protein. Upon cleavage of the first 11 amino acids, the recombinant protein displays essentially the same substrate specificity as observed earlier for the native plant enzyme. Product formation of the newly designed, truncated recombinant enzyme is consistent with light‐induced accumulation of methylated flavonoid conjugates in the ice plant. Therefore, substrate affinity and regiospecificity of an O‐methyltransferase in vivo and in vitro can be controlled by cleavage of an N‐terminal domain.

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg2+-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg2+-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.

Betalains replace the anthocyanins in flowers and fruits of plants of most families of the Caryophyllales. Unexpectedly, they were also found in some higher fungi. Whereas the anthocyanin-analogous functions of betalains in flower and fruit colouration are obvious, their role in fungi remains obscure. The nature of newly identified betalains as well as final structure elucidation of earlier putatively described compounds published within the last decade is compiled in this report. Recent advances in research on betalain biosynthesis is also covered, including description of some ‘early’ reactions, i.e. betalain-specific dopa formation in plants and fungi and extradiolic dopa cleavage in fungi. Work on betalain-specific glucosyltransferases (GTs) has given new insights into the evolution of secondary plant enzymes. It is proposed that these GTs are phylogenetically related to flavonoid GTs. It was found that the decisive steps in betalain biosynthesis, i.e. condensation of the betalain chromophore betalamic acid with cyclo-dopa and amino acids or amines in the respective aldimine formation of the red-violet betacyanins and the yellow betaxanthins, are most likely to be non-enzymatic. Betalains have attracted workers in applied fields because of their use for food colouring and their antioxidant and radical scavenging properties for protection against certain oxidative stress-related disorders.This review describes structure elucidation of betalains published within the last decade. Recent advances in betalain biosynthesis are also covered, i.e. enzymatic steps of ‘early’ (dopa formation) and ‘late’ reactions (glucosylation and acylation) as well as non-enzymatic steps (cyclo-dopa and aldimine formation).

IntroductionA Summary of Bioactive Glycosylated Secondary MetabolitesAgents that Interact with DNAEnediynesBleomycinsDiazobenzofluorenesAnthracyclinesPluramycinsAureolic AcidsAgents that Interact with RNAOrthosomycinsMacrolidesAminoglycosidesAmicetinsAgents that Interact with Cell Walls and Cell MembranesNon‐Ribosomal PeptidesPolyenesSaccharomicinsAgents that Interact with ProteinsIndolocarbazolesCoumarinsBenzoisochromanequinonesAvermectinsAngucyclinesCardiac GlycosidesLignansAnthraquinone GlycosidesGinsenosidesGlycoalkaloidsGlucosinolatesAgents that Interact with Other (or Undefined) TargetsPlant PhenolicsMono‐ and Triterpenoid GlycosidesPlant Polymeric Natural GlycosidesConclusionsReferences

Betanidin 6-O-glucosyltransferase (6-GT) is involved in the glycosylation of betacyanins, which replace the chromogenic anthocyanins as flower colorants in the Caryophyllales. The 6-GT cDNA was cloned from a cDNA library of Dorotheanthus bellidiformis (Burm. f.) N.E. Br., and the amino acid and nucleotide sequences were shown to be distinctly different from the corresponding betanidin 5-O-glucosyltransferase (5-GT) from the same plant species. Although both enzymes share very similar substrates, the proteins show only 19% amino acid sequence identity. In contrast, the protein sequence of the 6-GT showed significant identity to GTs from other species and may identify a new cluster of putative anthocyanidin GTs. Therefore, 6-GT and 5-GT apparently have evolved independently from ancestral glucosyltransferases involved in flavonoid biosynthesis.

Mesembryanthemum crystallinum L. (Aizoaceae) is a drought‐ and salt‐tolerant halophyte that is able to endure harsh environmental conditions. Upon irradiation with high light irradiance (1200–1500 µ mol m−2 s−1) it displays a rapid cell‐specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought treatment. The accumulation of these compounds, the betacyanins and acylated flavonol glycosides, increases if the plants are exposed to polychromatic radiation with a progressively decreasing short‐wave cut‐off in the ultraviolet range. The response is localized in the epidermal bladder cells on the tips of young leaves and epidermal layers of fully expanded leaves. It is demonstrated that the accumulation of flavonols and betacyanins can be described by a weakly sigmoid dose function in combination with an exponential decrease of the response function of the plant with increasing wavelength.

Plants are exposed to a wide range of toxic and bioactive low-molecular-weight molecules from both exogenous and endogenous sources. Glycosylation is one of the primary sedative mechanisms that plants utilise in order to maintain metabolic homeostasis. Recently, a range of glycosyltransferases has been characterized in detail with regard to substrate specificity. The next step in increasing our understanding of the biology of glycosylation will require information regarding the exact role of individual glycosyltransferases in planta, as well as an insight into their potential involvement in metabolon-complexes. Hopefully, this will answer how a large number of glycosyltransferases with broad, rather than narrow, substrate specificity can be constrained in order to avoid interfering with other pathways of primary and secondary metabolism. These and other topics are discussed.