Biochemical characterization of anArabidopsis glucosyltransferase with high activity toward Jasmonic acid

Biochemical characterization of the recombinant gene products from theArabidopsis glucosyltransferase multigene family has identified one enzyme with high activity toward the plant cellular regulator jasmonic acid (JA). The protein, AtJGT1 (UDP-glucose:JA glucosyltransferase), also has significant activities with other substrates, such as dihydrojasmonicacid, indole-3-acetic acid (IAA), indole-3-propionic acid, and indole-3-butyric acid. TheK _M values of AtJGT1 for JA or IAA are similar to those of anArabidopsis IAA glucosyltransferase UGT84B1 previously reported. Northern blot analysis showed thatAtJGTI is highly expressed in the leaves, but only slightly detectable in the roots, stems, and inflorescences. This study describes the first biochemical analysis of a recombinant glucosyltransferase with JA activity, and provides the foundation for future genetic approaches to understanding the role of JA-glucose inArabidopsis.     

In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria

Aims: To investigate the action of different polyphenolic compounds, extracted from red wine, grape marc and pine bark, on oral bacteria. Methods and Results: The anti-microbial activity of extracts was examined by determining the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration using the macro dilution broth technique. Their effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. The effect on glucosyltransferase activity was analysed through the reductions in the overall reaction rate and the quantity of insoluble glucan (ISG) synthesized. Pine bark and grape marc extracts were the most effective inhibitors of the multi-species biofilm formation and of the ISG synthesis. Conclusion: The tested components showed an interesting anti-plaque activity in vitro. Significance and Impact of the Study: This is, to our knowledge, the first and the most complete report on the properties of wine and pine bark extracts that could be used for oral disease prevention purpose.     

Cloning,extracellular expression and characterization of a predominant β-CGTase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. G1 in <Emphasis Type="Italic">E. coli</Emphasis>

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.     

Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae

We reported previously that a recombinant salicylic acid (SA) glucosyltransferase1 (AtSGT1) from Arabidopsis thaliana catalyzes the formation of both SA 2-O-beta-D-glucoside (SAG) and the glucose ester of SA (SGE). Here, transgenic Arabidopsis plants overexpressing AtSGT1 have been constructed, and their phenotypes analyzed. Compared to wild-type plants, transgenic plants showed an increased susceptibility to Pseudomonas syringae and reduced the accumulation levels of both free SA and its glucosylated forms (SAG and SGE). On the other hand, the overexpression increased the levels of methyl salicylate (MeSA) and methyl salicylate 2-O-beta-D-glucoside (MeSAG), and also induced SA carboxyl methyltransferase1 (AtBSMT1) expression, whose products catalyze the conversion of SA to MeSA. Our data indicate that reduced resistance by AtSGT1 overexpression results from a reduction in SA content, which is at least in part caused by increases in MeSAG and MeSA levels at the expense of SA. Our study also suggests that genetic manipulation of AtSGT1 can be utilized as an important regulatory tool for pathogen control.     

Two glucosyltransferases are involved in detoxification of benzoxazinoids in maize

Benzoxazinoids are major compounds involved in chemical defence in grasses. These toxins are stored in the vacuole as glucosides. Two glucosyltransferases, BX8 and BX9, that catalyse this last step of benzoxazinoid biosynthesis have been isolated via functional cloning. No close relative of these maize genes was found among the known glucosyltransferases. The enzymes display a very high degree of substrate specificity. DIMBOA, the major benzoxazinoid in young maize, is the preferred substrate. Both genes are highly expressed in young maize seedlings, the developmental stage with the highest activity of benzoxazinoid biosynthesis. Bx8 is included in the cluster of DIMBOA biosynthesis genes located on the short arm of chromosome 4. Hence, the gene cluster comprises three different enzymatic functions and a complete set of genes for the biosynthesis of DIBOA glucoside. Bx9 mapped to chromosome 1. Expression of Bx8 and Bx9 in Arabidopsis corroborated the potency of the enzymes in detoxification of their substrates. This capacity might have implications for allelopathic interactions.     

Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148

An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Controlling the association of adamantyl-substituted poly{N-[tris(hydroxymethyl)methyl]acrylamide} and a beta-cyclodextrin/epichlorohydrin polymer by a small drug molecule--naproxen

Two polymeric substances, a poly{N-[tris(hydroxymethyl)methyl]acrylamide} (THMMA) substituted with adamantyl moieties and a beta-cyclodextrin/epichlorohydrin polycondensate, formed a host-guest type complex, which resulted in the gel formation upon mixing of these two compounds at appropriate conditions. Introduction of a drug molecule, i.e., naproxen, that was able to fill the beta-cyclodextrin cavities, thus expulsing adamantyl moieties, led to disruption of such association and inhibition of gel formation. The conditions required for the association of the two polymeric components and formation of the gel, as well as the dynamics of its inhibition by addition of naproxen was established. The procedure of using solutions of two associating polymers and an appropriate drug competitor can be used at targeted viscosupplementation.     

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Plants emit a variety of volatiles in response to herbivore attack, and (Z)‐3‐hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)‐3‐hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)‐3‐hexenol remained unknown until today. In this study, uridine‐diphosphate‐dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)‐3‐hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53‐1 and UGT85A53‐2 showed the highest activity towards (Z)‐3‐hexenol and are distinct from UGT85A53‐3, which displayed a similar catalytic efficiency for (Z)‐3‐hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)‐3‐hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53‐1 in tobacco significantly increased the level of (Z)‐3‐hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)‐3‐hexenol UGT not only provides the foundation for the biotechnological production of (Z)‐3‐hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.     

γ-Cyclodextrin: a review on enzymatic production and applications

Cyclodextrins are cyclic α-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are α-, β-, and γ-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of γ-cyclodextrin as well as its difference with α- and β-cyclodextrins. As all known wild-type CGTases produce a mixture of α-, β-, and γ-cyclodextrins, the obtaining of a CGTase predominantly producing γ-cyclodextrin is discussed. Recently, more economic production processes for γ-cyclodextrin have been developed using improved γ-CGTases and appropriate complexing agents. Compared with α- and β-cyclodextrins, γ-cyclodextrin has a larger internal cavity, higher water solubility, and more bioavailability, so it has wider applications in many industries, especially in the food and pharmaceutical industries.