Search

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A “one-pot” system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A "one-pot" system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated S N 2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1 H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology. CtMaT1, a plant malonyltransferase with both unprecedented substrate promiscuity and malonylation regiospecificity, was functionally and structurally identified, and employed in universal enzymatic synthesis of diverse malonylated glycosides with potential druggabilities. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: The BAHD (benzylalcohol O-acetyl transferase, anthocyanin O-hydroxycinnamoyl transferase, N-hydroxycinnamoyl anthranilate benzoyl transferase and deacetylvindoline 4-O-acetyltransferase), has various biological functions in plants, including catalyzing the biosynthesis of terpenes, phenolics and esters, participating in plant stress response, affecting cell stability, and regulating fruit quality. Methods: Bioinformatics methods, real-time fluorescence quantitative PCR technology, and ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer were used to explore the relationship between the BAHD gene family and malonyl ginsenosides in Panax ginseng. Results: In this study, 103 BAHD genes were identified in P. ginseng, mainly distributed in three major clades. Most PgBAHDs contain cis-acting elements associated with abiotic stress response and plant hormone response. Among the 103 genes, 68 PgBAHDs are WGD (whole-genome duplication) genes. The significance of malonylation in biosynthesis has garnered considerable attention in the study of malonyltransferases. The phylogenetic tree results showed 34 PgBAHDs were clustered with genes that have malonyl characterization. Among them, seven PgBAHDs (PgBAHD4, 45, 65, 74, 90, 97, and 99) showed correlations > 0.9 with crucial enzyme genes involved in ginsenoside biosynthesis and > 0.8 with malonyl ginsenosides. These seven genes were considered potential candidates involved in the biosynthesis of malonyl ginsenosides. Discussion: These results help elucidate the structure, evolution, and functions of the P. ginseng BAHD gene family, and establish the foundation for further research on the mechanism of BAHD genes in ginsenoside biosynthesis. [ABSTRACT FROM AUTHOR]

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl‐CoA‐dependent enzyme homologous to xenobiotic metabolizing arylamine N‐acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N‐malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N‐acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel‐like" entries separated by a "bridge‐like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N‐terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl‐CoA better than malonyl‐CoA, dimerization changes the active site to allow malonyl‐CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl‐group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl‐group, acetyl‐CoA cannot form such stabilizing interactions, while longer acyl‐CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control. [ABSTRACT FROM AUTHOR]

SUMMARY: In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening‐related, receptacle‐specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated. Significance Statement: FaMYB123 is a fruit‐specific transcription factor whose expression is regulated by the two major hormones related to the control of strawberry (Fragaria × ananassa) ripening, ABA and auxins. FaMYB123‐RNAi fruits showed its implication in the final steps of anthocyanin/flavonoid biosynthesis, revealing a newly identified malonyltransferase, FaMT1. The metabolite analysis showed a concomitant decrease in the contents of total anthocyanin and flavonoid, particularly malonyl derivates. Furthermore, FaMYB123 physically interacts with FabHLH3, and together they transactivate FaMT1 expression. [ABSTRACT FROM AUTHOR]

Summary: Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin‐based polyacylated anthocyanins. However, recent breeding programs developed several red‐flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high‐performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin‐based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red‐flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6‐O‐glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol‐6‐O‐(6′‐O‐malonyl)‐glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3‐O‐glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell‐free protein expression system and assayed. We determined that a UDP‐glucose‐dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red‐flowered gentians via copigmentation effects. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer (HGT) is a well-documented evolutionary driving phenomenon in prokaryotes and eukaryotes, but its impact on the plant kingdom has remained elusive. A recent study provides compelling evidences, which support the idea that a plant-derived gene allows for the detoxification of plant defense metabolites in a polyphagous arthropod herbivore. [ABSTRACT FROM AUTHOR]

Introduction: The BAHD (benzylalcohol O-acetyl transferase, anthocyanin O-hydroxycinnamoyl transferase, N-hydroxycinnamoyl anthranilate benzoyl transferase and deacetylvindoline 4-O-acetyltransferase), has various biological functions in plants, including catalyzing the biosynthesis of terpenes, phenolics and esters, participating in plant stress response, affecting cell stability, and regulating fruit quality., Methods: Bioinformatics methods, real-time fluorescence quantitative PCR technology, and ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer were used to explore the relationship between the BAHD gene family and malonyl ginsenosides in Panax ginseng ., Results: In this study, 103 BAHD genes were identified in P. ginseng , mainly distributed in three major clades. Most PgBAHDs contain cis-acting elements associated with abiotic stress response and plant hormone response. Among the 103 genes, 68 PgBAHDs are WGD (whole-genome duplication) genes. The significance of malonylation in biosynthesis has garnered considerable attention in the study of malonyltransferases. The phylogenetic tree results showed 34 PgBAHDs were clustered with genes that have malonyl characterization. Among them, seven PgBAHDs ( PgBAHD4 , 45 , 65 , 74 , 90 , 97 , and 99 ) showed correlations > 0.9 with crucial enzyme genes involved in ginsenoside biosynthesis and > 0.8 with malonyl ginsenosides. These seven genes were considered potential candidates involved in the biosynthesis of malonyl ginsenosides., Discussion: These results help elucidate the structure, evolution, and functions of the P. ginseng BAHD gene family, and establish the foundation for further research on the mechanism of BAHD genes in ginsenoside biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Yan, Yan, Piao, Wang, Lei, Yang, Zhang, Li, Di and Yang.)

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure–function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.

Malonylated isoflavones are the major forms of isoflavonoids in soybean plants, the genes responsible for their biosyntheses are not well understood, nor their physiological functions. Here we report a new benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase (BAHD) family isoflavone glucoside malonyltransferase GmIMaT1, and GmIMaT3, which is allelic to the previously characterized GmMT7 and GmIF7MaT. Biochemical studies showed that recombinant GmIMaT1 and GmIMaT3 enzymes used malonyl-CoA and several isoflavone 7-O-glucosides as substrates. The Km values of GmIMaT1 for glycitin, genistin, and daidzin were 13.11, 23.04, and 36.28 μM, respectively, while these of GmIMaT3 were 12.94, 26.67, and 30.12 μM, respectively. Transgenic hairy roots overexpressing both GmIMaTs had increased levels of malonyldaidzin and malonylgenistin, and contents of daidzin and glycitin increased only in GmIMaT1-overexpression lines. The increased daidzein and genistein contents were detected only in GmIMaT3-overexpression lines. Knockdown of GmIMaT1 and GmIMaT3 reduced malonyldaidzin and malonylgenistin contents, and affected other isoflavonoids differently. GmIMaT1 is primarily localized to the endoplasmic reticulum while GmIMaT3 is primarily in the cytosol. By examining their transcript changes corresponding to the altered isoflavone metabolic profiles under various environmental and hormonal stresses, we probed the possible functions of GmIMaTs. Two GmIMaTs displayed distinct tissue expression patterns and respond differently to various factors in modifying isoflavone 7-O-glucosides under various stresses.

Malonylated isoflavones are the major forms of isoflavonoids in soybean plants, the genes responsible for their biosyntheses are not well understood, nor their physiological functions. Here we report a new benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase (BAHD) family isoflavone glucoside malonyltransferase GmIMaT1, and GmIMaT3, which is allelic to the previously characterized GmMT7 and GmIF7MaT. Biochemical studies showed that recombinant GmIMaT1 and GmIMaT3 enzymes used malonyl-CoA and several isoflavone 7-Oglucosides as substrates. The Km values of GmIMaT1 for glycitin, genistin, and daidzin were 13.11, 23.04, and 36.28 mM, respectively, while these of GmIMaT3 were 12.94, 26.67, and 30.12 mM, respectively. Transgenic hairy roots overexpressing both GmIMaTs had increased levels of malonyldaidzin and malonylgenistin, and contents of daidzin and glycitin increased only in GmIMaT1-overexpression lines. The increased daidzein and genistein contents were detected only in GmIMaT3-overexpression lines. Knockdown of GmIMaT1 and GmIMaT3 reduced malonyldaidzin and malonylgenistin contents, and affected other isoflavonoids differently. GmIMaT1 is primarily localized to the endoplasmic reticulum while GmIMaT3 is primarily in the cytosol. By examining their transcript changes corresponding to the altered isoflavone metabolic profiles under various environmental and hormonal stresses, we probed the possible functions of GmIMaTs. Two GmIMaTs displayed distinct tissue expression patterns and respond differently to various factors in modifying isoflavone 7-O-glucosides under various stresses. [ABSTRACT FROM AUTHOR]

Tobacco cells ( Nicotiana tabacum L.) accumulate harmful naphthols in the form of malonylated glucosides ( Taguchi et al., 2005 ). Here, we showed that the malonylation of glucosides is a system to metabolize xenobiotics and is common to higher plants. Moreover, some plantlets including Arabidopsis thaliana excreted some of the incorporated naphthols into the culture media as their glucosides. In order to analyze the function of malonylation in the metabolism of these xenobiotics, we identified a malonyltransferase gene ( At5g39050) responsible for the malonylation of these compounds in A. thaliana. The recombinant enzyme had malonyltransferase activity toward several phenolic glucosides including naphthol glucosides. A knockout mutant of At5g39050 ( pmat1) exposed to naphthols accumulated only a few malonylglucosides in the cell, and released larger amounts of simple glucosides into the culture medium. In contrast, forced expression of At5g39050 in the pmat1 mutant resulted in increased malonylglucoside accumulation and decreased glucoside excretion to the media. The results provided clear evidence of whether the release of glucosides or the storage of malonylglucosides was determined by the At5g39050 expression level. A similar event in naphthol metabolism was observed in the tobacco mutant with a suppressed malonyltransferase gene ( NtMaT1). These results suggested that malonylation could be a key reaction to separate the way of xenobiotics disposition, that is, release from cell surface or storage in vacuoles. [ABSTRACT FROM AUTHOR]

(Iso)flavonoids are commonly accumulated as malonylated or acetylated glycoconjugates in legumes. Sequence analysis on EST database of the model legume Medicago truncatula enabled us to identify nine cDNA sequences encoding BAHD super-family enzymes that are distinct from the most of the characterized anthocyanin/flavonol acyltransferase genes in other species. Functional characterization revealed that three of these corresponding enzymes, MtMaT1, 2 and 3, specifically recognize malonyl CoA as an acyl donor and catalyze the malonylation of a range of isoflavone 7- O-glucosides in vitro. These malonyltransferase genes displayed distinct tissue-specific expression patterns and responded differentially to biotic and abiotic stresses. Consistent with gene expression, the level of the accumulated malonyl isoflavone glucoside was altered in the roots of M. truncatula grown under normal and drought-stressed conditions. Overexpression of the MtMaT1 gene in a previously engineered Arabidopsis line that accumulates genistein glycosides ( Proc. Natl Acad. Sci. USA, 99, 2002:14578) led to a malonylated product. Confocal microscopy of the transiently expressed MtMaT1–GFP fusion revealed strong fluorescence in both the cytoplasm and nucleus of M. truncatula and tobacco leaf cells. A truncated MtMaT1 lacking the C-terminal polypeptide of 110 amino acid residues that include the DFGWG motif, the single conserved sequence signature of BAHD super-family members, retained considerable catalytic efficiency, but showed an altered optimum pH preference for maximum activity. Such C-terminal polypeptide deletion or deletion of the DFGWG motif alone led to improper folding of the transiently expressed GFP fusion protein in living cells, and impaired nuclear localization of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: A simple and versatile method for the chemical synthesis of 21-hydroxypregnane 21-O-malonyl hemiesters which may be important intermediates of cardenolide biosynthesis is described. Starting from commercial β-methyldigitoxin, acid hydrolysis followed by 3β-O-acetylation and ozonolysis with reductive cleavage of the ozonides afforded 3β-acetoxy-5β-pregnane-14β,21-diol-20-one which was finally converted into the target compound by treatment with malonyl chloride. The malonylation protocol was optimized using deoxycorticosterone (DOC) as the pregnane educt. [Copyright &y& Elsevier]

Isoflavonoids are a diverse group of biologically active natural products that accumulate in soybean seeds during development. The majority of isoflavonoids are accumulated in the form of their glyco- and malonyl-conjugates in soybean seeds. The conjugation step confers stability and solubility to isoflavone aglycones enabling their compartmentalization to vacuoles or transport to the site of accumulation. A functional genomic approach was used to identify isoflavonoid specific glycosyltransferase (UGT) and malonyltransferase (MT) from soybean (Glycine max) seeds. An expressed sequence tag database for soybean was searched by key words to make a list of candidate genes. The full-length cDNAs for candidate UGTs and MTs were obtained and cloned into an expression vector for the production of recombinant enzymes. The in vitro enzymatic activity assays were conducted for recombinant UGTs and MTs using uridine diphosphate glucose and malonyl CoA, respectively, as donors with isoflavone substrates. Among several recombinant enzymes, UGT73F2 showed glycosylation activity towards all three soybean isoflavone aglycones and GmMT7 exhibited malonylation activity towards isoflavone glycosides. The subcellular localization study revealed both UGT73F2 and GmMT7 to be in the cytoplasm. The transcripts and protein accumulation patterns for UGT73F2 and GmMT7 genes have provided further support for their in planta function. [ABSTRACT FROM PUBLISHER]

Abstract: A cDNA from soybean (Glycine max (L.) Merr.), GmIF7MaT, encoding malonyl-CoA:isoflavone 7-O-glucoside-6″-O-malonyltransferase, was cloned and characterized. Soybeans produce large amounts of isoflavones, which primarily accumulate in the form of their 7-O-(6″-O-malonyl-β-d-glucosides). The cDNA was obtained by a homology-based strategy for the cDNA cloning of some flavonoid glucoside-specific malonyltransferases of the BAHD family. The expressed gene product, GmIF7MaT, efficiently catalyzed specific malonyl transfer reactions from malonyl-CoA to isoflavone 7-O-β-d-glucosides yielding the corresponding isoflavone 7-O-(6″-O-malonyl-β-d-glucosides) (IF7MaT activity). The k cat values of GmIF7MaT were much greater than those of other flavonoid glucoside-specific malonyltransferases with their preferred substrates, while the K m values were at comparable levels. GmIF7MaT was expressed in the roots of G. max seedlings more abundantly than in hypocotyl and cotyledon. Native IF7MaT activity was also observed in the roots, suggesting that GmIF7MaT is involved in the biosynthesis from isoflavone 7-O-β-d-glucosides to the corresponding isoflavone 7-O-(6″-O-malonyl-β-d-glucosides) in G. max. This protein is a member of flavonoid glucoside-specific acyltransferases in the BAHD family. [Copyright &y& Elsevier]

Summary: 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)-β-glucoside which is one of the precursors of ternatins, respectively. Moreover, WM accumulates minor delphinidin glycosides – 3-O-β-glucoside, 3-O-(2″-O-α-rhamnosyl)-β-glucoside, 3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-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-α-rhamnosyl)-β-glucoside, 3-O-(6″-O-α-rhamnosyl)-β-glucoside, 3-O-(2″,6″-di-O-α-rhamnosyl)-β-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-β-glucoside in the presence of malonyl-CoA, it also has a preference for other anthocyanidin 3-O-β-glucosides. It could use flavonol 3-O-β-glucosides in much lower specific activities than anthocyanins; however, it could not utilize 3-O-(2″-O-α-rhamnosyl)-β-glucosides of anthocyanins and flavonols, and 3,3′-di- and 3,3′,5′-tri-O-β-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-β-glucoside. The crude enzymes prepared from WM and WW had the same enzymatic characteristics. These results suggested that 3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucosides of flavonoids were synthesized via 3-O-(6″-O-malonyl)-β-glucosides rather than via 3-O-(2″-O-α-rhamnosyl)-β-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-β-glucosides. [Copyright &y& Elsevier]

Tobacco cells (Nicotiana tabacumL. Bright Yellow T-13) exposed to harmful naphthols accumulate them as glucosylated and further modified compounds [Taguchiet al.(2003a)Plant Sci. 164, 231–240]. In this study, we identified the accumulated compounds to be 6′-O-malonylated glucosides of naphthols. Cells treated with various phenolic compounds accumulated the flavonoids mainly as malonylglucosides. To clarify the function of this malonylation in tobacco, we isolated the cDNA encoding a malonyltransferase (NtMaT1) from a cDNA library derived from tobacco cells. The heterologous expression of the gene inEscherichia colirevealed that the recombinant enzyme had malonyltransferase activity against several phenolic glucosides such as flavonoid 7-O-glucosides, flavonoid 3-O-glucosides and naphthol glucosides. The substrate preference of the enzyme was similar to that of the tobacco cell extract. Malonylation activity in the transgenic cells markedly decreased with the suppression of the expression ofNtMaT1mRNA in tobacco BY-2 cells by RNA interference. The compounds administered to the transgenic cells were accumulated in the cells as glucosides or other modified compounds in place of malonylglucosides. These results show that NtMaT1 is the main catalyst of malonylation on glucosides of xenobiotic flavonoids and naphthols in tobacco plants. [ABSTRACT FROM AUTHOR]

In the flowers of chrysanthemum (Dendranthema×morifolium), cyanidin 3-O-3″,6″-O-dimalonylglucoside is accumulated as a dominant anthocyanin. In this study, we isolated two full-length cDNAs from chrysanthemum flowers, Dm3MaT1 and Dm3MaT2, coding for enzymes that should be responsible for malonylation steps involved in the biosynthesis of the pigment. Both cDNAs were heterologously expressed in Escherichia coli cells, and the expressed products were functionally characterized. The Dm3MaT1 cDNA coded for a protein of 459 amino acid residues with a calculated molecular weight of 51,224, which showed >70% of sequence identity to anthocyanidin 3-O-glucoside-6″-O-malonyltransferases of Dahlia variabilis and Senecio cruentus. Specificity analyses of Dm3MaT1 further confirmed its identity as an anthocyanidin 3-O-glucoside-6″-O-malonyltransferase, which exclusively catalyzed the malonyl-CoA-dependent malonyl transfer to anthocyanidin 3-O-glucoside to produce anthocyanidin 3-O-6″-O-malonylglucoside. On the other hand, the Dm3MaT2 cDNA coded for a protein of 460 amino acid residues with a calculated molecular weight of 51,710, showing identities of 93% (for the nucleotide sequence) and 89% (for the deduced amino acid sequence) to Dm3MaT1. Unlike Dm3MaT1, the recombinant Dm3MaT2 catalyzed the consecutive dimalonyl transfer to anthocyanidin 3-O-glucoside; it produced anthocyanidin 3-O-6″-O-malonylglucoside from anthocyanidin 3-O-glucoside and could subsequently produce anthocyanidin 3-O-3″,6″-O-dimalonylglucoside from anthocyanidin 3-O-6″-O-malonylglucoside. Thus, Dm3MaT2 was the anthocyanidin 3-O-glucoside-3″,6″-O-dimalonyltransferase and should be responsible for the second malonylation of anthocyanins in the flower, providing the first example of anthocyanin acyltransferase that can catalyze the consecutive malony transfer. [Copyright &y& Elsevier]

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants. [Copyright &y& Elsevier]

Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3-O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure–function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases. [ABSTRACT FROM AUTHOR]