Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula x Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.     

Structural characterization of the mitomycin 7-O-methyltransferase

Mitomycins are quinone‐containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin‐7‐O‐methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7‐O‐methylation of both C9β‐ and C9α‐configured 7‐hydroxymitomycins. We have determined the crystal structures of the MmcR–S‐adenosylhomocysteine (SAH) binary complex and MmcR–SAH–mitomycin A (MMA) ternary complex at resolutions of 1.9and 2.3 Å, respectively. The study revealed MmcR to adopt a common S‐adenosyl‐L ‐methionine‐dependent O‐methyltransferase fold and the presence of a structurally conserved active site general acid–base pair is consistent with a proton‐assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Altered regioselectivity of a poplar O-methyltransferase, POMT-7

O-Methylated flavonoids are biosynthesized by regioselective flavonoid O-methyltransferases (OMTs), which may account for the limited number of naturally occurring flavonoids in nature. It was previously shown that poplar POMT-7 regioselectively methylates the 7-hydroxyl group of flavones, whereas rice ROMT-9 regioselectively methylates the 3'-hydroxyl group of the substrate. We co-expressed both OMT genes (POMT-7 and ROMT-9) in E. coli and carried out biotransformation experiments of some flavonoids with the transformed E. coli strain. Contrast to the predicted regioselectivity of both POMT-7 and ROMT-9, unexpected methylation reaction products, i.e. 3',4'-O-methylated flavonoids, in addition to the predicted ones, were obtained with luteolin (5,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) as substrates. Reactions using the 3'-O-methyl derivative of luteolin and quercetin by POMT-7 revealed that the enzyme has altered its regioselectivity from the 7- to the 4'-hydroxyl groups. These results are discussed in terms of molecular modeling of POMT-7 in relation to its methyl donor.     

Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co²⁺ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.     

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine: vitexin 2"-O-rhamnoside 7-O-methyltransferase of Avena sativa L

An O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteine-sepharose. Km values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 microM and 15 microM, respectively. The lack of methyltransfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-O-methylvitexin 2"-O-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.     

Milbemycin C5-O-甲基转移酶基因的克隆

目的是克隆milbemycin C5-O-甲基转移酶基因。以阿维菌素（avermectin)产生菌S.avermitilis中C5－O－甲基转移酶基因aveD为探针,利用核酸杂交分析法将aveD的主要同源区定位在（22）g-8-E-10阳性克隆所整合的.3kb SatⅠ-BgⅢ片段上。最后对aveD同源区及附近的核苷酸序列进行了测序分析和相应区域的基因缺失分析。结果表明,该区域存在一个月与aveD同源性较强的基因milD。与aveD功能相似,milD参与milbemycin的生物合成,在milbemycin生物合成中可能催化C5位OH的甲基化反应。     

Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransferase

Caffeoyl coenzyme A 3-O-methyltransferases (CCoAOMTs) are S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) involved in lignin biosynthesis. Plant CCoAOMTs belong to a distinct family of OMTs, more closely related to the mammalian catechol OMTs than to other plant OMTs. The crystal structure of alfalfa (Medicago sativa) CCoAOMT in complex with the reaction products S-adenosine-l-homocysteine and feruloyl/sinapoyl CoAs presented here belong to a structurally and mechanistically distinct family of plant small molecule OMTs. These structures provide a new understanding of the substrate preferences and the catalytic mechanism accompanying CCoAOMT-mediated O-methylation of CoA-linked phenylpropanoid substrates.     

Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.     

Beta-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum

S-Adenosyl-L-methionine:beta-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of beta-peltatin in position 6 thus forming beta-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 degrees C. The enzyme activity is strongly inhibited by MnSO(4), FeCl(3), FeSO(4) and ZnSO(4) as well as S-adenosyl-homocysteine. Mg(2+) and EDTA did not influence the methylation of beta-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and beta-peltatin and apparent K(m)-values of 15 microM and 40 microM, respectively, were determined for these substrates. Substrate inhibition was observed for beta-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:beta-peltatin 6-O-methyltransferase. Highest specific activities of beta-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.     

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis

Darapladib is one of the most potent Lp-PLA₂ (Lipoprotein-associated phospholipase A₂) inhibitor with an IC₅₀ of 0.25?nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA₂.     

Nucleocytoplasmic-localized acyltransferases catalyze the malonylation of 7-O-glycosidic (iso)flavones in Medicago truncatula

(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.     

Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase

Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (approximately 20%) and modest increase in cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.     

Regioselectivity of lipases in organic solvents

Summary A facile system was developed for the quantitative determination of lipase regioselectivities in organic solvents towards the 1(3)-position of glycerides. It was utilized for the measurement of the regioselectivities displayed by lipase preparations fromMucor miehei (Lipozyme),Pseudomonas fluorescens andRhizopus delemar. It was shown that the lipases fromMucor miehei andPseudomonas fluorescens do in fact not display the high 1(3)-specifities reported in the literature for these enzymes.     

Bioactive flavones of Sida cordifolia

Two new flavones: 5,7-dihydroxy-3-isoprenyl flavone (1) and 5-hydroxy-3-isoprenyl flavone (2), along with two known compounds β-sitosterol and stigmasterol have been isolated from the chloroform extract of Sida cordifolia. Their structures were established on the basis of spectroscopic analysis. Flavones were tested for their analgesic and anti-inflammatory activity.