A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 '-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 '-O-glucosides through the use of whole-cell biocatalysis.     

Biosynthesis of malonylated flavonoid glycosides on the basis of malonyltransferase activity in the petals of Clitoria ternatea

The crude malonyltransferase from the petals of Clitoria ternatea was characterized enzymatically to investigate its role on the biosynthetic pathways of anthocyanins and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB) and mauve flower variety (WM) accumulate polyacylated anthocyanins (ternatins) and delphinidin 3-O-(6'-O-malonyl)-beta-glucoside which is one of the precursors of ternatins, respectively. Moreover, WM accumulates minor delphinidin glycosides - 3-O-beta-glucoside, 3-O-(2'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2'-O-alpha-rhamnosyl-6'-O-malonyl)-beta-glucoside of delphinidin. These glycosidic patterns for minor anthocyanins in WM are also found among the minor flavonol glycosides in all the varieties including a white flower variety (WW) although the major flavonol glycosides are 3-O-(2'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(6'-O-alpha-rhamnosyl)-beta-glucoside, 3-O-(2',6'-di-O-alpha-rhamnosyl)-beta-glucoside of kaempferol, quercetin, and myricetin. How do the enzymatic characteristics affect the variety of glycosidic patterns in the flavonoid glycoside biosynthesis among these varieties? While the enzyme from DB highly preferred delphinidin 3-O-beta-glucoside in the presence of malonyl-CoA, it also has a preference for other anthocyanidin 3-O-beta-glucosides. It could use flavonol 3-O-beta-glucosides in much lower specific activities than anthocyanins; however, it could not utilize 3-O-(2'-O-alpha-rhamnosyl)-beta-glucosides of anthocyanins and flavonols, and 3,3'-di- and 3,3',5'-tri-O-beta-glucoside of delphinidin - other possible precursors in ternatins biosynthesis. It highly preferred malonyl-CoA as an acyl donor in the presence of delphinidin 3-O-beta-glucoside. The crude enzymes prepared from WM and WW had the same enzymatic characteristics. These results suggested that 3-O-(2'-O-alpha-rhamnosyl-6'-O-malonyl)-beta-glucosides of flavonoids were synthesized via 3-O-(6'-O-malonyl)-beta-glucosides rather than via 3-O-(2'-O-alpha-rhamnosyl)-beta-glucosides, and that malonylation proceeded prior to glucosylation at the B-ring of delphinidin in the early biosynthetic steps towards ternatins. It seemed that the substrate specificities largely affected the difference in the accumulated amount of malonylated glycosides between anthocyanins and flavonols although they are not simply proportional to the accumulation ratio. This enzyme might join in the production of both malonylanthocyanins and flavonol malonylglycosides as a result of broad substrate specificities towards flavonoid 3-O-beta-glucosides.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle

In an effort to characterize fruit ripening-related genes functionally, two glucosyltransferases, FaGT6 and FaGT7, were cloned from a strawberry (Fragaria x ananassa) cDNA library and the full-length open reading frames were amplified by rapid amplification of cDNA ends. FaGT6 and FaGT7 were expressed heterologously as fusion proteins in Escherichia coli and target protein was purified using affinity chromatography. Both recombinant enzymes exhibited a broad substrate tolerance in vitro, accepting numerous flavonoids, hydroxycoumarins, and naphthols. FaGT6 formed 3-O-glucosides and minor amounts of 7-O-, 4'-O-, and 3'-O-monoglucosides and one diglucoside from flavonols such as quercetin. FaGT7 converted quercetin to the 3-O-glucoside and 4'-O-glucoside and minor levels of the 7- and 3'-isomers but formed no diglucoside. Gene expression studies showed that both genes are strongly expressed in achenes of small-sized green fruits, while the expression levels were generally lower in the receptacle. Significant levels of quercetin 3-O-, 7-O-, and 4'-O-glucosides, kaempferol 3-O- and 7-O-glucosides, as well as isorhamnetin 7-O-glucoside, were identified in achenes and the receptacle. In the receptacle, the expression of both genes is negatively controlled by auxin which correlates with the ripening-related gene expression in this tissue. Salicylic acid, a known signal molecule in plant defence, induces the expression of both genes. Thus, it appears that FaGT6 and FaGT7 are involved in the glucosylation of flavonols and may also participate in xenobiotic metabolism. The latter function is supported by the proven ability of strawberries to glucosylate selected unnatural substrates injected in ripe fruits. This report presents the first biochemical characterization of enzymes mainly expressed in strawberry achenes and provides the foundation of flavonoid metabolism in the seeds.     

Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds

The development of efficient tools is required for the eco-friendly detoxification and effective detection of neurotoxic organophosphates (OPs). Although enzymes have received significant attention as biocatalysts because of their high specific activity, the uneconomic and labor-intensive processes of enzyme production and purification make their broad use in practical applications difficult. Because whole-cell systems offer several advantages compared with free enzymes, including high stability, a reduced purification requirement, and low preparation cost, they have been suggested as promising biocatalysts for the detoxification and detection of OPs. To develop efficient whole-cell biocatalysts with enhanced activity and a broad spectrum of substrate specificity, several factors have been considered, namely the selected strains, the chosen OP-hydrolyzing enzymes, where enzymes are localized in a cell, and which enhancer will assist the expression, function, and folding of the enzyme. In this article, we review the current investigative progress in the development of engineered whole-cell biocatalysts with excellent OP-hydrolyzing activity, a broad spectrum of substrate specificity, and outstanding stability for the detoxification and detection of OPs.     

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.     

Regioselective synthesis of plant (iso)flavone glycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.     

Purification and Identification of Flavonoids from the Yellow Green Cocoon Shell (Sasamayu) of the Silkworm,Bombyx mori

Three quercetin glycosides, quercetin 5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, and quercetin 4′-O-β-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-β-D-glucoside and kaempferol 7-O-β-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.     

Production of a novel quercetin glycoside through metabolic engineering of Escherichia coli

Most flavonoids exist as sugar conjugates. Naturally occurring flavonoid sugar conjugates include glucose, galactose, glucuronide, rhamnose, xylose, and arabinose. These flavonoid glycosides have diverse physiological activities, depending on the type of sugar attached. To synthesize an unnatural flavonoid glycoside, Actinobacillus actinomycetemcomitans gene tll (encoding dTDP-6-deoxy-L-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-L-mannose to dTDP-6-deoxytalose) was introduced into Escherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases (UGTs) were screened to find a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain of E. coli with quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-deoxytalose) by increasing the supplement of dTDP-6-deoxytalose in E. coli, we engineered nucleotide biosynthetic genes of E. coli, such as galU (UTP-glucose 1-phosphate uridyltransferase), rffA (dTDP-4-oxo-6-deoxy-d-glucose transaminase), and/or rfbD (dTDP-4-dehydrorahmnose reductase). The engineered E. coli strain produced approximately 98 mg of quercetin 3-O-(6-deoxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose and quercetin 3-O-rhamnose, were also significantly reduced.     

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.     

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C‐glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C‐glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C‐glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C‐glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C‐glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C‐glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over‐expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.     

微生物糖基转移酶催化合成槲皮素糖苷及其抗炎活性评价

利用微生物来源的糖基转移酶GT-1、GT-2和BTGT-1对槲皮素进行糖基化修饰。研究发现这3种酶均能够催化以槲皮素和UDP-葡萄糖为底物生成槲皮素-3-O-β-D-葡萄糖（异槲皮苷）,GT-1、 GT-2和BTGT-1催化反应的产物生成率分别为5.33%、15.18%和63.82%。通过对槲皮素、异槲皮苷和槲皮素-N-乙酰-D-氨基葡萄糖进行水溶性以及体外细胞抗炎活性检测,发现槲皮素经糖基化后其水溶性得到较大提高,异槲皮苷和槲皮素-N-乙酰-D-氨基葡萄糖水溶性分别是槲皮素的13.8倍和15.4倍。同等浓度下槲皮素糖苷对RAW264.7 细胞的毒性作用低于槲皮素,且3种化合物在一定浓度范围内对LPS诱导RAW264.7细胞释放NO、 IL-1β、IL-6 都有显著的抑制作用,且呈剂量依赖性,研究表明3种化合物都具有一定的抗炎作用。     

Flavonoid characterization and in vitro antioxidant activity of Aconitum anthora L. (Ranunculaceae)

In this paper, we report studies on morphological, phytochemical, and biological aspects of a population belonging to Aconitum anthora L. Two compounds, quercetin 3-O-((beta-D-glucopyranosyl-(1-->3)-(4-O-(E-p-coumaroyl))-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside))-7-O-alpha-L-rhamnopyranoside (1) and kaempferol 3-O-((beta-D-glucopyranosyl-(1-->3)-(4-O-(E-p-coumaroyl))-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside))-7-O-alpha-L-rhamnopyranoside (2), together with two known flavonol glycosides (3-4) were isolated and identified from A. anthora. The antioxidant activity of the four identified flavonoids was screened by three in vitro tests.     

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-β-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-β-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-β-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-β-D-glucopyranoside (18), were biosynthesized by the hairy roots.     

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Three flavonol glycosides, kaempferol 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, quercetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, and myricetin 3-O-(2",6"-di-O-alpha-rhamnosyl)-beta-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2(G)- rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.     

一种作用于花青素和甜菊醇的甜菊糖基转移酶的基因克隆和功能分析

本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53.2K Da。与常见的糖基转移酶基因比较,相似性达44%以上,且具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。     

一种作用于花青素和甜菊醇的甜菊糖基转移酶的基因克隆和功能分析

本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53．2KDa。与常见的糖基转移酶基因比较,相似性达44％以上,工具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。     

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.     

Flavonoid <Emphasis Type="Italic">O</Emphasis>-Diglucosyltransferase from Rice: Molecular Cloning and Characterization

Among more than 100 rice uridine diphosphate glycosyltransferases (UGTs), OsUGT-3 was selected as a candidate for producing flavonoid O-diglycosyltransferases based on phylogenetic analysis and molecular docking. This gene was functionally expressed in Escherichia coli. Analysis of kaempferol, luteolin, quercetin, and tricin reaction products using liquid chromatography-mass spectrometry revealed that these were diglucosylated. The glucosylation positions of kaempferol, which was the best substrate, were determined to be the 3- and 7-hydroxyl groups. This is the first flavonoid O-diglucosyltransferase described from rice.