Enzymes of phosphatidylcholine synthesis in lemna, soybean, and carrot

Cell-free extracts from Lemna and suspension cultured carrot (Daucus Carota L.) catalyze S-adenosylmethionine-dependent N-methylations of phosphoethanolamine, phosphomethylethanolamine, and phosphodimethylethanolamine; extracts of suspension cultured soybean (Glycine max), of phosphoethanolamine only. Material pelleted from each tissue between 15,000 and 100,000g catalyzes S-adenosylmethionine-dependent N-methylations of phosphatidylmethylethanolamine and phosphatidyl-dimethylethanolamine, but not phosphatidylethanolamine. Extracts from each tissue catalyze CTP-dependent cytidylyltransfers to each of the three methylated phosphoethanolamine derivatives, forming the corresponding CDP derivatives. Some of the properties of the activities investigated are reported. On the basis of in vivo labeling experiments, we have proposed (AH Datko, SH Mudd 1988 Plant Physiol 88: 854-861) differing pathways for phosphatidylcholine synthesis in which, after a common committing step, N-methylation of phosphoethanolamine, subsequent methylations occur in Lemna almost exclusively at the phospho-base level; in soybean, at the phosphatidyl-base level; and in carrot, at both levels. Thus, among the activities investigated, at least those required for the operation of the proposed pathways have been positively demonstrated. The extent to which the present results explain the differences between these pathways is discussed, and a speculation offered as to how these differences may have arisen phylogenetically.     

Phosphatidylcholine synthesis: differing patterns in soybean and carrot

The methylation steps in the biosynthesis of phosphatidylcholine by tissue culture preparations of carrot (Daucus carota L.) and soybean (Glycine max), and by soybean leaf discs, have been studied. Preparations were incubated with tracer concentrations of l-[³H₃C]methionine and the kinetics of appearance of radioactivity in phosphomethylethanolamine, phosphodimethylethanolamine, phosphocholine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, methylethanolamine, dimethylethanolamine, and choline followed at short incubation times. With soybean (tissue culture or leaves), an initial methylation utilizes phosphoethanolamine as substrate, forming phosphomethylethanolamine. The latter is converted to phosphatidylmethylethanolamine, which is successively methylated to phosphatidyldimethyethanolamine and to phosphatidylcholine. With carrot, again, an initial methylation is of phosphoethanolamine. Subsequent methylations occur at both the phospho-base and phosphatidyl-base levels. Both of these patterns differ qualitatively from that previously demonstrated in Lemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in which all three methylations occur at the phospho-base level. For soybean and carrot, some added contribution from initial methylation of phosphatidylethanolamine has not been excluded. These results, together with those from similar experiments carried out with water-stressed barley leaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814-822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 Plant Physiol 71: 692-700) suggest that in higher plants some, perhaps all, phosphatidylcholine synthesis occurs via a common committing step (conversion of phosphoethanolamine to phosphomethylethanolamine) followed by a methylation pattern which differs from plant to plant.     

Phosphocholine synthesis in spinach: Characterization of phosphoethanolamine N-methyltransferase

Phosphocholine is a precursor for phosphatidylcholine or it may be hydrolysed to choline. Choline can be oxidized to form the compatible osmolyte glycine betaine which is accumulated by many plants under conditions of osmotic stress. In Spinacia oleracea phosphocholine is synthesized by 3 sequential N‐methylations of phosphoethanolamine with the first step catalysed by the enzyme phosphoethanolamine N‐methyltransferase (EC 2.1.1.103). This enzyme has been partially purified 5400‐fold from spinach leaves using a combination of ammonium sulphate fractionation, followed by chromatographic separations on DEAE‐Sepharose, phenyl‐Sepharose, Ω‐aminohexyl‐agarose, Mono Q and adenosine‐agarose. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation and silver‐staining of the final preparation revealed several polypeptides present, only one of which with an estimated molecular mass of 54 kDa could be photoaffinity cross‐linked to the substrate [³H] S‐adenosyl‐l ‐methionine. HPLC gel permeation chromatography was used to obtain an estimate for the native molecular mass of 77 kDa. Enzyme activity was optimal at pH 7.8 in HEPES‐KOH buffer, it was inhibited by S‐adenosyl‐l ‐homocysteine, phosphocholine, phosphate, Mn²⁺ and Co²⁺ but not by ethanolamine, methylethanolamine, dimethylethanolamine, choline, glycine betaine or Mg²⁺. Using phosphoethanolamine as substrate, the final preparation had a specific activity of 189 nmol mg^?1 protein min^?1. The reaction products were identified and their relative abundance estimated following separation by TLC as phosphomethylethanolamine (87%), phosphodimethylethanolamine (10%) and phosphocholine (2%). Thus, a highly purified preparation of phosphoethanolamine N‐methyltransferase was shown to catalyse 3 successive N‐methylations of phosphoethanolamine. Photoaffinity cross‐linking of proteins extracted from leaves of spinach followed by SDS‐PAGE and autoradiography shows that a 54‐kDa radiolabelled polypeptide was more prominent in extracts from salinized plants and barely visible in extracts from plants exposed to prolonged dark periods, a pattern which corresponds to the salt and light‐responsive changes in phosphoethanolamine N‐methylating activity. Thus, the production of phosphocholine for glycine betaine accumulation in spinach can be mediated by a single phosphobase N‐methyltransferase which is more abundant in salt‐stressed plants.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana

Arabidopsis possesses several genes encoding aspartate aminotransferase, which catalyzes the bidirectional conversion of aspartate into glutamate. These amino acids together with asparagine and glutamine play an important role in N storage and distribution. In addition, they act as precursors for other amino acids. The gene encoding cytosolic aspartate aminotransferase, Asp2, was found to be induced upon infection with the necrotrophic pathogen Botrytis cinerea in Arabidopsis. Asp2 over-expression lines and a T-DNA insertion mutant were used to study the role of aspartate aminotransferase in Arabidopsis defence responses. Over-expression of Asp2 led to changes in aspartate content and aspartate-derived amino acids. The Asp2 knockout mutant was also slightly affected in its amino acid composition. Under standard growth conditions, the Asp2 transgenic lines did not show morphological changes in comparison with the wild-type. However, transgenic lines with the highest Asp2 expression displayed more spreading lesions when infected with B. cinerea. We discuss how this gene involved in amino acid metabolism might interact with plant defence responses.     

Cloning and functional characterization of a caffeic acid O-methyltransferase from Trigonella foenum-graecum L

A cDNA encoding an O-methyltransferase (namely FGCOMT1) was identified from the medicinal plant Trigonella foenum-graecum L. The FGCOMT1 enzyme is a functional caffeic acid O-methyltransferase (COMT) and is localized in the cytosol. Kinetic analysis indicated that FGCOMT1 protein exhibited the highest catalyzing efficiency towards 5-hydroxy ferulic acid and caffeic acid as substrates, but did not possess the abilities to methylate either quercetin or tricetin in vitro. Furthermore, transformation of Arabidopsis loss-of-function Atomt1 mutant with a FGCOMT1 cDNA partially complements accumulation of sinapoyl derivatives but did not function to produce the major methylated flavonol isorhamnetin in seeds. The results from this study indicated that FGCOMT1 is a COMT with substrate preference to monomeric lignin precursors but is not involved in the flavonoid methylation in T. foenum-graecum L.     

Identification of clones containing DNA complementary to phenylethanolamine N-methyltransferase mRNA

Specific poly(A)mRNA for phenylethanolamine N-methyltransferase was isolated from bovine adrenal medulla by immunoprecipitation of polysomal mRNA with antibodies to bovine adrenal phenylethanolamine N-methyltransferase. Antibody-polysome complexes were recovered by Protein A Sepharose affinity chromatography. Phenylethanolamine N-methyltransferase mRNA, enriched 50-fold as judged by quantitative immunoprecipitation of translation products, was used as a template for the synthesis of complementary DNA (cDNA). Double-stranded cDNA was tailed with deoxycytosine and inserted into the Pst 1 site of poly(dG)-tailed plasmid pBR322. The resultant recombinant plasmids were used to transform competent E. coli strain 294. Tetracycline-resistant ampicillin-sensitive clones were screened by positive hybridization selection, and preliminary screening identified 2 out of 36 clones containing phenylethanolamine N-methyltransferase cDNA inserts. One phenylethanolamine N-methyltransferase cDNA insert was isolated from the plasmid DNA by digestion with Pst 1 and was found to be approximately 350 base-pairs in length. Northern blot analysis revealed that this phenylethanolamine N-methyltransferase cDNA probe strongly hybridized to an RNA species of approximately 1100 nucleotides.     

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.     

Molecular cloning and characterization of 5-enolpyruvylshikimate-3-phosphate synthase gene from Convolvulus arvensis L.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, catalyzes an essential step in the shikimate pathway for aromatic amino acid biosynthesis. The full-length cDNA of 1,751 nucleotides (CaEPSPS, Genbank accession number: EU698030) from Convolvulus arvensis was cloned and characterized. The CaEPSPS encodes a polypeptide of 520 amino acids with a calculated molecular weight of 55.5 kDa and an isoelectric point of 7.05. The results of homology analysis revealed that CaEPSPS showed highly homologous with EPSPS proteins from other plant species. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in stems, leaves and roots, with lower expression in roots. CaEPSPS expression level could increase significantly with glyphosate treatment, and reached its maximum at 24 h after glyphosate application. We fused CaEPSPS to the CaMV 35S promoter and introduced the chimeric gene into Arabidopsis. The resultant expression of CaEPSPS in transgenic Arabidopsis plants exhibited enhanced tolerance to glyphosate in comparison with control.     

Human Indolethylamine N-Methyltransferase: cDNA Cloning and Expression, Gene Cloning, and Chromosomal Localization

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K_m value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon–intron splice junctions conformed to the “GT-AG” rule, and no canonical TATA or CAAT sequences were present within the 5′-flanking region of the gene. Human INMT mapped to chromosome 7p15.2–p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Molecular cloning and characterization of the gene encoding N′-[(5′-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (BBM II) isomerase from Arabidopsis thaliana

We have isolated an Arabidopsis BBM II isomerase cDNA from an Arabidopsis cDNA library, by means of functional complementation of the E. coli hisA mutant strain HfrG6. The isolated cDNA encodes a polypeptide of 304 amino acids with a calculated molecular weight of 33?363. Sequence comparison with the HIS6 proteins of yeasts revealed that Arabidopsis BBM II isomerase contains an N-terminal extension of approximately 40 amino acids that shows the general properties of chloroplast transit peptides. This finding is consistent with the localization of other histidine biosynthetic enzymes, such as imidazoleglycerolphosphate dehydratase and histidinol dehydrogenase, in the chloroplasts in higher plants. The primary structure of the mature protein was 50% and 42% identical, respectively, to the HIS6 proteins of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, while no prominent sequence similarity to the bacterial BBM II isomerase was found. That the isolated Arabidopsis cDNA actually encodes a functionally active BBM II isomerase activity was confirmed in an in vitro enzyme assay using a crude extract prepared from strain HfrG6 transformed with the Arabidopsis BBM II isomerase cDNA.     

Molecular cloning and expression of tryptophan decarboxylase from Mitragyna speciosa

Tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine in mitragynine biosynthesis via the shikimate pathway. Using the rapid amplification of cDNA ends (RACE) technique, the gene encoding TDC from Mitragyna speciosa was cloned (designated as MsTDC). The MsTDC cDNA contained an open reading frame (ORF) of 1,521 base pairs (bp) encoding 506 amino acid residues. It had a pyridoxal-phosphate (PLP)-binding site at the amino acid position 313–334 residues. The MsTDC showed homology of 68–76 % to the TDC of other plants. Heterologous expression in Escherichia coli afforded the soluble proteins as an apparent band of 57 kDa as judged by SDS-PAGE. Expression of the MsTDC in M. speciosa hairy roots under the 35S promoter was performed by insertion of MsTDC into pCAMBIA1300-gfp. The transgenic hairy root lines were detected by fluorescence microscopy and showed an increased accumulation of tryptamine.     

cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme

The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT, EC ). A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without ethanolamine- or phosphatidylethanolamine N-methyltransferase activity. The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplication and fusion. Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants. Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer. PEAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine. Monomethyl- and dimethylphosphoethanolamine are detected as reaction intermediates. A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation. Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.     

The reaction mechanism of phenylethanolamine N-methyltransferase: A density functional theory study

Hybrid density functional theory methods were used to investigate the reaction mechanism of human phenylethanolamine N-methyltransferase (hPNMT). This enzyme catalyzes the S-adenosyl-l-methionine-dependent conversion of norepinephrine to epinephrine, which constitutes the terminal step in the catecholamine biosynthesis. Several models of the active site were constructed based on the X-ray structure. Geometries of the stationary points along the reaction path were optimized and the reaction barrier and energy were calculated and compared to the experimental values. The calculations demonstrate that the reaction takes place via an S_N2 mechanism with methyl transfer being rate-limiting, a suggestion supported by mutagenesis studies. Optimal agreement with experimental data is reached using a model in which both active site glutamates are protonated. Overall, the mechanism of hPNMT is more similar to those of catechol O-methyltransferase and glycine N-methyltransferase than to that of guanidinoacetate N-methyltransferase in which methyl transfer is coupled to proton transfer.