Comparative Analysis of the Extradiol Ring-Cleavage Dioxygenase LigB from Arabidopsis and 3,4-Dihydroxyphenylalanine Dioxygenase from Betalain-Producing Plants.

Diverse arrays of naturally occurring compounds in plants are synthesized by specialized metabolic enzymes, many of which are distributed taxonomically. Although anthocyanin pigments are widely distributed and ubiquitous, betalains have replaced anthocyanins in most families in Caryophyllales. Anthocyanins and betalains never occur together in the same plant. The formation of betalamic acid, catalyzed by 3,4-dihydroxyphenylalanine (DOPA) 4,5-extradiol dioxygenase (DOD), is a key step in betalain biosynthesis. DODs in betalain-producing plants are coded by LigB genes, homologs of which have been identified in a wide range of higher plant orders, as well as in certain fungi and bacteria. Two classes of LigB homologs have been reported: those found in anthocyanin-producing species and those found in betalain-producing species, which contain DOD. To gain insight into the evolution of specialized metabolic enzymes involved in betalain biosynthesis, we performed a comparative biochemical analysis of Arabidopsis LigB, an extradiol ring-cleavage dioxygenase in anthocyanin-producing Arabidopsis and Phytolacca DOD1 of betalain-producing Phytolacca americana. We show that Arabidopsis LigB catalyzes 2,3-extradiol cleavage of DOPA to synthesize muscaflavin, whereas Phytolacca DOD1 converts DOPA to betalamic acid via 4,5-extradiol cleavage. Arabidopsis LigB also converts caffeic acid, a ubiquitous phenolic compound in higher plants, to iso-arabidopic acid in vitro via 2,3-extradiol cleavage of the aromatic ring. Amino-acid substitution in Arabidopsis LigB and Phytolacca DOD1 led to variable extradiol ring-cleavage function, supporting the suggestion that catalytic promiscuity serves as a starting point for the divergence of new enzymatic activities.

Crown-ether-mediated crystal structures of the glycosyltransferase PaGT3 from Phytolacca americana.

Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structure-function relationship of PaGT3, its crystal structure was determined. The sugar-donor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptor-binding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein. The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.

An Ambidextrous Polyphenol Glycosyltransferase PaGT2 from Phytolacca americana.

The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.

Optical Resolution of (RS)-Denopamine to (R)-Denopamine P-D- Glucoside by Glucosyltransferase from Phytolacca americana Expressed in Recombinant Escherichia coli.

The optical resolution of racemic compounds by stereoselective glucosylation was investigated using plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. The glucosyltransferase glucosylated chemoselectively the phenolic hydroxyl group of phenol compounds. The (R)-stereoselective glucosylation of (RS)-denopamine by glucosyltransferase occurred to give (R)-denopamine ß-D-glucoside.

Structural analysis of a type 1 ribosome inactivating protein reveals multiple L­asparagine­N­acetyl­D­glucosamine monosaccharide modifications: Implications for cytotoxicity.

Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome­inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin­ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X­ray crystal structure of PAP­S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP­S1aci shares ~95% sequence identity with PAP­S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP­S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP­S1aci, which has no effect on catalytic Glu175 positioning or overall active­site topology, yet appears to come at the expense of strained main­chain geometry at the pre­proline residue Val173. Notably, a rare type of N­glycosylation was detected consisting of N­acetyl­D­glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP­S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N­glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA­binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N­glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor­mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

Enzymatic Synthesis of Quercetin Monoglucopyranoside and Maltooligosaccharides.

Quercetin 3-O-ß-monoglucopyranoside and quercetin 3-O-ß-maltooligosaccharide were synthesized from quercetin using glucosyltransferase-3 from Phytolacca americana and cyclodextrin glucanotransferase.

Synthesis of Resveratrol Glycosides by Plant Glucosyltransferase and Cyclodextrin Glucanotransferase and Their Neuroprotective Activity.

Resveratrol was converted by glucosyltransferase from Phytolacca americana into its 3- and 4'-O-ß-D-glucosides. On the other hand, further glycosylation of resveratrol 4'-O-ß-D-glucoside by cyclodextrin glucanotransferase gave the 4'-O-ß-maltoside, 4'-O-ß-maltotrioside, 4'-O-ß-maltotetraoside, and 4'-O-ß- maltopentaoside of resveratrol. The six resveratrol glycosides synthesized here showed higher phosphodiesterase inhibitory activity than resveratrol.

Synthesis of ε-Viniferin Glycosides by Glucosyltransferase from Phytolacca americana and their Inhibitory Activity on Histamine Release from Rat Peritoneal Mast Cells.

Glycosylation of (+)-ε-viniferin was investigated using glucosyltransferase from Phytolacca americana (PaGT3) as a biocatalyst. (+)-ε-Viniferin was converted by PaGT3 into its 4b- and 13b-ß-D-glucosides, the inhibitory activities on histamine release from rat peritoneal mast cells of which were higher than that of (+)-ε-viniferin.

Comparative analysis of two DOPA dioxygenases from Phytolacca Americana.

The comparative analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis was carried out. The recombinant protein of PaDOD catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. The role of DOD genes is discussed in the evolutionary context using phylogenetic analysis, suggesting that DOD might have been duplicated early in evolution and that accumulation of base substitutions could have led to the different characteristics of DODs within the betalain-producing Caryophyllales.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Synthesis of 3,5,3',4'-tetrahydroxy-trans-stilbene-4'-O-beta-D-glucopyranoside by glucosyltransferases from Phytolacca americana.

Two glucosyltransferase isozymes from Phytolacca americana, PaGT3 and PaGT2, catalyzed stereo- and regio-selective monoglucosylation of 3,5,3',4'-tetrahydroxy-trans-stilbene to yield 3,5,3',4'-tetrahydroxy-trans-stilbene-4'-O-beta-D-glucopyranoside.

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana.

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-ß-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-ß-glucoside: 3-O-ß-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

[Antioxidative enzymes play key roles in cadmium tolerance of Phytolacca americana].

Phytolacca americana L. has the capacity to take up and accumulate to very high levels heavy metals such as Mn and Cd, and is used for phytoextraction of heavy metal contaminated soils. The role of antioxidative enzyme of Phytolacca americana in response to Cd stress is unknown. The 6-week-old seedlings of Phytolacca americana were exposed to half strength Hoagland solution with 200 micromol/L CdCl2 or 400 micromol/L CdCl2 for 4 days. The content of H2O2 and MDA, and electrolyte leakage increased, while the photosynthetic rate decreased, indicated that the oxidative damage induced by Cd stress in Phytolacca americana was one of the metal toxicity mechanism. The activities of SOD and POD increased rapidly with elevated Cd concentration and exposure time, CAT activity was stable in response to 200 micromol/L CdCl2 stress, and increased only at 3 d later upon 400 micromol/L CdCl2, treatment. Suggested that the enzymatic antioxidation capacity played important role in Cd tolerance of hyperaccumulator plant.

Isolation and expression analysis of two DOPA dioxygenases in Phytolacca americana.

Betacyanins and anthocyanins, two main red flower pigments, never occur together in the same plant. Although the anthocyanin biosynthetic pathway has been well analyzed, the biosynthetic genes and the regulatory mechanism of the betacyanin biosynthesis are still obscure. We cloned two cDNAs of DOPA dioxygenase from Phytolacca americana, PaDOD1 and PaDOD2, that may be involved in the betalain biosynthesis. The deduced amino acid sequence of PaDOD1 and PaDOD2 showed approximately 80% homology to each other. The promoter regions of PaDOD1 and PaDOD2 were isolated by inverse PCR and analyzed using PLACE database. Some putative MYB, bHLH, and environmental stress-responsive transcription factor binding sites were detected in the PaDOD1 and PaDOD2 promoter regions. Expression patterns of PaDOD1 and PaDOD2 in suspension cultures of P. americana were investigated by semiquantitative RT-PCR. The transcripts of PaDODs were found in both betacyanin-producing red cells and non-betacyanin-producing white cells, suggesting that not only the expression of DOD, but also the supplementation of DOPA might be a regulatory step for the betalain biosynthesis in P. americana.

Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species.

Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. There has been no report so far to find anthocyanins and betacyanins existing together within the same plant. This curious phenomenon has been examined from genetic and evolutionary perspectives, however nothing is known at the molecular level about the mutual exclusion of anthocyanins and betacyanins in higher plants. Here, we show that spinach (Spinacia oleracea) and pokeweed (Phytolacca americana), which are both members of the Caryophyllales, have functional anthocyanidin synthases (ANSs). The ability of ANSs of the Caryophyllales to oxidize trans-leucocyanidin to cyanidin is comparable to that of ANSs in anthocyanin-producing plants. Expression profiles reveal that, in spinach, dihydroflavonol 4-reductase (DFR) and ANS are not expressed in most tissues and organs, except seeds, in which ANS may contribute to proanthocyanidin synthesis. One possible explanation for the lack of anthocyanins in the Caryophyllales is the suppression or limited expression of the DFR and ANS.