cDNA Cloning of PhosphoethanolamineN-Methyltransferase from Spinach by Complementation inSchizosaccharomyces pombe and Characterization of the Recombinant Enzyme

Nuccio, Michael L.; Ziemak, Michael J.; Henry, Susan A.; Weretilnyk, Elizabeth A.; Hanson, Andrew D.

doi:10.1074/jbc.275.19.14095

Cited by 101 publications

(145 citation statements)

References 36 publications

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“…Similarly, orcinol OMT was reported to carry out two sequential O-methylations in rose (Rosa 3 hybrida) scent biosynthesis (Lavid et al, 2002;Scalliet et al, 2002). Sequential methylation has also been reported for certain N-methyltransferases (Nuccio et al, 2000;Charron et al, 2002), but these proteins possess a different structural domain. In comparison, SOMT1 accepted both benzylisoquinoline and protoberberine substrates, showed no discrimination toward N-desmethyl or N-methylated BIAs, and was able to catalyze sequential O-methylations on both the isoquinoline and benzyl moieties.…”

Section: Benzylisoquinoline (Rs)-norlaudanosoline Was Not Accepted mentioning

confidence: 99%

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

2012

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Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatographytandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-Omethyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9-and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 39-and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy.

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Section: Benzylisoquinoline (Rs)-norlaudanosoline Was Not Accepted mentioning

confidence: 99%

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

2012

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“…Blots were hybridized overnight at 65°C in 5ϫ SSPE͞5ϫ Denhardt's solution͞0.5% SDS͞100 g⅐ml Ϫ1 fragmented salmon sperm DNA and washed at 37°C in 0.1ϫ SSPE containing 1% SDS (standard saline phosphate͞EDTA: 0.75 M NaCl͞0.05 M phosphate, pH 7.4͞5 mM EDTA. Blots were probed with the PCR-amplified coding sequence of LeGCHI and then stripped and reprobed with an 18S rRNA sequence (27). Probes were labeled with 32 P by random priming.…”

Section: Methodsmentioning

confidence: 99%

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Basset

Quinlivan

Ziemak

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of Ϸ26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying Ϸ50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.T etrahydrofolate and its derivatives (folates) are essential cofactors for one-carbon transfer reactions in all organisms. Similar to bacteria and yeasts, plants make folates de novo from pterin, p-aminobenzoate (PABA), and glutamate moieties (1, 2). In contrast, humans and other mammals lack a complete folatesynthesis pathway and thus need dietary folate. Because plant foods are major folate sources, and folate deficiency is a global health problem, enhancing plant folate content is a prime target for metabolic engineering (1, 3). This engineering demands knowledge of the biosynthetic pathway.The plant folate-synthesis pathway is not understood fully, but is most probably similar to that in bacteria (1, 2). The pterin hydroxymethyldihydropteroate is formed from GTP, and PABA from chorismate. The pterin and PABA units are condensed, glutamylated, and reduced to give tetrahydrofolate, and a polyglutamyl tail is added (4). Plant genes and enzymes for the final five steps have been characterized (5, 6), and the enzymes have been shown to be mitochondrial (7,8). Far less is known for plants about the early steps that produce the pterin and PABA moieties.The first step of pterin synthesis is of special interest, because it commits GTP to pterin production and is considered to control flux into the pathway (9, 10). This step, mediated by GTP cyclohydrolase I (GC...

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“…The homology search revealed that a protein encoded by ORF1 is highly homologous to the EcGSMT (ϳ66% identity in amino acids) and N-terminal part of heterotrophic eubacterium A. halophila methyltransferase (ϳ61% identity in amino acids). The ORF1 protein showed some homology to the rat glycine N-methyltransferase (ϳ35% identity in amino acids) but essentially no homology to other methyltransferases including phosphoethanolamine N-methyltransferase (27). The protein encoded by ORF2 showed the highest homology to EcSDMT (ϳ52% identity in amino acids) and C-terminal part of A. halophila methyltransferase (ϳ49% identity in amino acids).…”

Section: Cloning Of Methyltransferase Genes From a Halophytica-mentioning

confidence: 91%

“…However, in yeast, these reactions are catalyzed by two enzymes, one mediating the first methylation of Ptd-EA and another mediating the last two (32). In the phospho-base pathway, presence of three separate N-methyltransferases has been reported in nerve tissues (33), whereas in plants, three-step methylation reactions were catalyzed by a single enzyme comprising two similar tandem methyltransferase domains (27). Thus, the biogenesis of Ntrimethyl compounds occurs via diversity methyltransferases.…”

Section: Discussionmentioning

confidence: 99%

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Isolation and Functional Characterization ofN-Methyltransferases That Catalyze Betaine Synthesis from Glycine in a Halotolerant Photosynthetic Organism Aphanothece halophytica

Waditee¹,

Tanaka²,

Aoki³

et al. 2003

Journal of Biological Chemistry

116

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Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. The most known biosynthetic pathways of betaine are the two-step oxidation of choline. Many bacteria, plants, and animals accumulate glycine betaine (here after betaine) under abiotic stress conditions (1-3). In these organisms, it was shown that betaine is synthesized by two steps, choline 3 betaine aldehyde 3 glycine betaine. The enzyme involved in the second step seems to be the same in plants, animals, and bacteria, namely NAD ϩ -dependent betaine-aldehyde dehydrogenase (4 -6). By contrast, different enzymes are involved for the first step. In plants, it was catalyzed by a novel Rieske-type iron-sulfur enzyme choline monooxygenase (7,8). In animals and many bacteria, the first step is catalyzed by membranebound choline dehydrogenase or soluble choline oxidase (9 -11). In some bacteria, choline dehydrogenase and choline oxidase also catalyze the second oxidation step (9 -11).It was suggested that betaine might be synthesized from glycine by a series of methylation reactions in archaebacterium Methanohalophilus portucalensis (12) and anaerobic phototrophic sulfur bacterium Ectothiorhodospira halochloris (13). Betaine synthesis from simple carbon sources has also been suggested in aerobic heterotrophic eubacterium Actinopolyspora halophila (13) and halotolerant cyanobacterium of Aphanothece halophytica (14). Recently, the methyltransferase genes that are involved in betaine synthesis have been isolated from E. halochloris and A. halophila (15). Two methyltransferase genes were involved in E. halochloris. One of gene products catalyzed the methylation reactions of glycine and sarcosine to sarcosine and dimethylglycine, respectively (EcGSMT), 1 whereas the other one catalyzed the methylations of sarcosine and dimethylglycine to dimethylglycine and betaine, respectively (EcSDMT) (15,16). By contrast, one ORF was found in A. halophila of which the N-and C-terminal parts had homologous sequences to those of EcGSMT and EcSDMT, respectively (15). The functionality of A. halophila methyltransferase was not well shown due to the formation of cell pellet when expressed in Escherichia coli.Glycine N-methyltransferase (GMT) catalyzing the methylation of glycine to sarcosine is known in mammalian cells although the enzymes catalyzing the further methylation steps do not occur (17,18). The homology of amino acid sequences between mammalian GMT and EcGSMT was low. No homologous sequences to those of EcGSMT, EcSDMT, and A. halophila methyltransferase could be found. Therefore, it was interesting to examine whether betaine is synthesized from glycine by three-step methylation reactions in other organisms.

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cDNA Cloning of PhosphoethanolamineN-Methyltransferase from Spinach by Complementation inSchizosaccharomyces pombe and Characterization of the Recombinant Enzyme

Cited by 101 publications

References 36 publications

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Isolation and Functional Characterization ofN-Methyltransferases That Catalyze Betaine Synthesis from Glycine in a Halotolerant Photosynthetic Organism Aphanothece halophytica

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