Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

Alpha-tocopherol (vitamin E) is synthesized from gamma-tocopherol in chloroplasts by gamma-tocopherol methyltransferase (gamma-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of alpha-tocopherol, but are low in gamma-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene gamma-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allele (vte4-2) carrying a T-DNA insertion in the coding sequence of gamma-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of gamma-tocopherol accumulated, whereas alpha-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the gamma-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of alpha-tocopherol and gamma-tocopherol increased in wild type, and gamma-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that alpha-tocopherol can be replaced by gamma-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.     

Are there two DNA methyltransferase gene families in plant cells? A new potential methyltransferase gene isolated from an Arabidopsis thaliana genomic library.

Using the 1kb 3' terminal DNA fragment of the mouse methyltransferase cDNA as a probe and low stringent hybridisation conditions, a new potential methyltransferase (MTase) gene family was isolated from an Arabidopsis thaliana genomic DNA library. One clone (MTase-11), which gave the strongest signal at the Northern blot, was entirely sequenced (11483 bp) and further characterised. Under consideration of the likely open reading frames and our preliminary cDNA experiments we propose that the clone 11 gene encodes for an approximately 90 kD protein. As deduced form the DNA sequence this protein contains all conserved sequence motifs specific for the 5m cytosine MTases. MTase-11 gene expression was demonstrable in callus and during germination but not in one month old plants or in leaves.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Studies on S-adenosylmethionine–magnesium protoporphyrin methyltransferase in Euglena gracilis strain Z

1. An enzyme that methylates magnesium protoporphyrin was detected in extracts of light-grown and dark-grown cells of Euglena gracilis. The activity in light-grown cells is two to three times that in cells grown in the dark. 2. The activity is mainly located in the chloroplast fraction from light-grown cells and in proplastids in dark-grown cells. However, in cells grown either in the light or dark, about 15-20% is found in particle-free supernatant. 3. The chloroplast methylating enzyme was solubilized by the action of Tween 80 and partially purified. The properties were investigated. 4. From experiments in which etiolated cells were illuminated in the presence of inhibitors of chloroplast or cytoplasmic protein synthesis, it appears that the methylating enzyme is made on cytoplasmic ribosomes.     

Demonstration and solubilization of S-adenosylmethionine: γ-tocopherol methyltransferase from Capsicum chromoplasts

Through the use of Capsicum chromoplast membranes, we report for the first time the direct methylation of -tocopherol into -tocopherol in the presence of S-adenosylmethionine. Furthermore the S-adenosylmethionine: -tocopherol methyltransferase activity has been solubilized. On a protein basis, the activity recovered in the soluble preparation was higher than that bound to the membranes.     

Increased ��-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis ��-tocopherol methyltransferase

Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The ??-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. ??-tocopherol methyltransferase (??-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of ??- and ??-isomers to ??- and ??-isomers. In present study, the Arabidopsis ??-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the ??-/??-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the ??-isomers were converted to ??-isomers, especially the ??- and ??-tocotrienol levels were dramatically decreased. As a result, the ??-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the ??-tocotrienol content. The results showed that the ??-/??-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on ??-tocopherol level, which may reflect the fact that ??-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign ??-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world??s most important food crops.     

Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate     

S-Adenosyl-l-methionine–cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine-cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C(28) and C(29) sterol production in the algae.     

γ-Tocopherol methyltransferase from the green alga Chlamydomonas reinhardtii: functional characterization and expression analysis

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is a very important enzyme in tocopherol biosynthesis in all photosynthetic organisms. In this paper, we present the functional characterization and expression analysis of γ-TMT from the unicellular green alga Chlamydomonas reinhardtii. Recombinant TMT1 enzyme was purified and characterized. The size of TMT1 subunit was estimated as 37 kDa by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in accordance with the predicted molecular size after TMT1 cDNA sequence. Recombinant TMT1 also showed an apparent molecular mass of 37 kDa in its native conformation, suggesting that native TMT1 has a monomeric structure similar to the plant TMTs already characterized. pH and temperature dependence of TMT1 activity were also similar to plant TMTs. Substrate specificity studies showed that Chlamydomonas TMT1 is responsible for the conversion of γ- and δ-tocopherol to α- and β-tocopherol, respectively. The kinetic properties of Chlamydomonas recombinant γ-TMT activity were studied and γ-TMT1 has a similar affinity for γ- and δ-tocopherol. Promoter sequence analysis and expression analysis by northern blot revealed that tmt1 expression is strongly upregulated by high light and downregulated by low temperature. This regulatory pattern of tmt1 expression supports the idea that γ- and α-tocopherol play specific roles in the adaptation to growth under low temperature and high light stress conditions.     

Ultrastructural alterations induced by Δ(24(25))-sterol methyltransferase inhibitors on Trichomonas vaginalis

In a search for thermophilic ethanol-tolerant bacteria, water-sediment samples collected at springs in Yunnan province of China were screened by ethanol enrichment. A novel thermophilic bacterium, strain E13(T) , was isolated. It exhibits a unique and remarkable ability to preferably grow in the presence of ethanol and is able to tolerate 13% (v/v) ethanol at 60 °C. The isolate is a facultative aerobic, Gram-positive, motile, spore-forming rod that is capable of utilizing a range of carbon sources, such as xylose, arabinose and cellobiose. Phylogenetic analysis based on 16S rRNA gene similarity showed the strain to be affiliated with the species Anoxybacillus flavithermus (99.2% sequence similarity). DNA-DNA hybridization comparisons demonstrated a 64.8% DNA-DNA relatedness between strain E13(T) and A. flavithermus DSM 2641(T) . On the basis of phenotypic characteristics, phylogenetic data and DNA-DNA hybridization data, it was concluded that the isolate merited classification as a novel subspecies of A. flavithermus, for which the name Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. is proposed. The type strain of this subspecies is E13(T) (=CCTCC AB2010187(T) =KCTC 13759(T) ).     

Protein arginine methyltransferase 6 enhances ligand-dependent and -independent activity of estrogen receptor α via distinct mechanisms

Recent studies reported that protein arginine methyltransferase 6 (PRMT6) enhances estrogen-induced activity of estrogen receptor α (ERα) and dysfunction of PRMT6 is associated with overall better survival for ERα-positive breast cancer patients. However, it is unclear how PRMT6 promotes ERα activity. Here we report that PRMT6 specifically interacts with ERα at its ligand-binding domain. PRMT6 also methylates ERα both in vitro and in vivo. In addition to enhancing estrogen-induced ERα activity, PRMT6 over-expression up-regulates estrogen-independent activity of ERα and PRMT6 gene silencing in MCF7 cells inhibits ligand-independent ERα activation. More interestingly, the effect of PRMT6 on the ligand-independent ERα activity does not require its methyltransferase activity. Instead, PRMT6 competes with Hsp90 for ERα binding: PRMT6 and Hsp90 bindings to ERα are mutually exclusive and PRMT6 over-expression reduces ERα interaction with Hsp90. In conclusion, PRMT6 requires its methyltransferase activity to enhance ERα's ligand-induced activity, but its effect on ligand-independent activity is likely mediated through competing with Hsp90 for binding to the C-terminal domain of ERα. PRMT6-ERα interaction would prevent ERα-Hsp90 association. Since Hsp90 and associated chaperones serve to maintain ERα conformation for ligand-binding yet functionally inactive, inhibition of ERα-Hsp90 interaction would relieve ERα from the constraint of chaperone complex.     

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T₂ seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.     

Do damaged proteins accumulate in Caenorhabditis elegans L-isoaspartate methyltransferase (pcm-1) deletion mutants?

The protein l-isoaspartate (d-aspartate) O-methyltransferase (E.C. 2. 1.1.77) can initiate the conversion of isomerized and racemized aspartyl residues to their normal l-aspartyl forms and has therefore been hypothesized to function as a repair enzyme, responsible for helping to limit the accumulation of damaged proteins in aging organisms. In this study, the effect of a disruption in the pcm-1 gene encoding the l-isoaspartyl methyltransferase was investigated in the nematode Caenorhabditis elegans. It was found that damaged proteins recognized by this enzyme accumulated to significant levels during long-term incubation of both pcm-1+ and pcm-1- nematodes in a specialized larval stage called the dauer. The l-isoaspartyl methyltransferase-deficient mutants accumulated about twice the level of damaged proteins as the control nematodes during dauer aging. The mutants also accumulated higher levels of damage when both strains were incubated at 30 degrees C for up to 3 days. However, when nonviable nematodes were removed in a Percoll separation, similar levels of damage were measured between the two strains following both dauer aging and 30 degrees C incubation. Both strains were able to effectively eliminate damaged proteins recognized by the methyltransferase after recovery from dauer. Characterization of the methyl-accepting polypeptide substrates which accumulate in aged dauers revealed that although substrates of all molecular weights are present, the majority of substrates are peptides not precipitated by acetone. These results suggest that protein degradation, rather than repair, may be the major mechanism by which C. elegans eliminates damaged proteins containing l-isoaspartyl residues.     

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa)

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.     

Structure–activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1

A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK_a, were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK_a of the quinoline unit had little apparent influence on activity.