Dependent Methyltransferase Research Articles

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosyl-l-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.

Molecular Architecture of a C-3′-Methyltransferase Involved in the Biosynthesis of d-Tetronitrose

S-Adenosylmethionine (SAM)-dependent methyltransferases are involved in a myriad of biological processes, including signal transduction, chromatin repair, metabolism, and biosyntheses, among others. Here we report the high-resolution structure of a novel C-3'-methyltransferase involved in the production of D-tetronitrose, an unusual sugar found attached to the antitumor agent tetrocarcin A or the antibiotic kijanimicin. Specifically, this enzyme, referred to as TcaB9 and cloned from Micromonospora chalcea, catalyzes the conversion of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose to dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-D-glucose. For this analysis, two structures were determined to 1.5 A resolution: one in which the enzyme was crystallized in the presence of SAM and dTMP and the other with the protein complexed to S-adenosylhomocysteine and its dTDP-linked sugar product. The overall fold of the monomeric enzyme can be described in terms of three domains. The N-terminal domain harbors the binding site for a zinc ion that is ligated by four cysteines. The middle domain adopts the canonical "SAM-binding" fold with a seven-stranded mixed beta-sheet flanked on either side by three alpha-helices. This domain is responsible for anchoring the SAM cofactor to the protein. Strikingly, the C-terminal domain also contains a seven-stranded beta-sheet, and it appears to be related to the middle domain by an approximate 2-fold rotational axis, thus suggesting TcaB9 arose via gene duplication. Key residues involved in sugar binding include His 181, Glu 224, His 225, and Tyr 222. Their possible roles in catalysis are discussed.

Genistein mediates perturbations in one‐carbon metabolism during diet‐induced folate deficiency

Populations consuming isoflavone‐rich diets exhibit lower prevalence of heart disease and estrogen‐responsive cancers. Genistein, a17β‐estradiol‐like isoflavone, has been shown to modify the fetal epigenome in utero in the agouti mouse model and modulate DNA methylation patterns in estrogen‐responsive tissues. Moreover, it also exhibits antiproliferative action in cancerous cells via suppression of S‐adenosylmethionine –dependent DNA methyltransferases, leading to site‐specific hypomethylation. However, it is not known if genistein modulates one‐carbon metabolism, a process that when disrupted leads to dysregulation of the epigenome. The objective of this study was to characterize the metabolic actions of genistein during moderate dietary folate deficiency. Male Sprague Dawley ratswere fed an ad lib control or folate‐deficient (FD) diet with or without genistein supplementation (300 mg/kg diet) for 8 wk. Plasma homocysteine (Hcy) concentrations in genistein‐supplemented, FD animals (16.7 μM) was lower (P = 0.02) than the Hcy concentration of FD animals not supplemented with genistein (27.9 μM). Hepatic enzyme analysis suggests the ability of genistein to attenuate hyperhomocysteinemia in FD rats is not linked to diminished Hcy production. Clearly, genistein has the ability to modulate Hcy and methyl‐group metabolism; however, the underlying mechanisms remain unknown.Grant Funding Source: AHA assn

Enzyme-Catalyzed Transfer of a Ketone Group from an S-Adenosylmethionine Analogue: A Tool for the Functional Analysis of Methyltransferases

S-adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analogue functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analogue displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analogue expands the toolbox available to interrogate the biochemical functions of methyltransferases.

Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

Crystal structure ofStreptococcus pneumoniaeSp1610, a putative tRNA methyltransferase, in complex withS‐adenosyl‐L‐methionine

Streptococcus pneumoniae Sp1610, a Class-I fold S-adenosylmethionine (AdoMet)-dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N(1)-adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand-free and the AdoMet-bound forms at resolutions of 2.0 and 3.0 A, respectively. The protein is organized into two structural domains: the N-terminal catalytic domain with a Class I AdoMet-dependent methyltransferase fold, and the C-terminal substrate recognition domain with a novel fold of four alpha-helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.

Synthesis of S‐Adenosyl‐L‐homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Hyperhomocysteinemia is Associated with Decreased Erythropoietin Expression in Rats

Background/Aims: Elevated plasma homocysteine (Hcy) levels have been identified as a pathogenic factor causing a variety of pathological changes in different cells and tissues. In vertebrates, Hcy is produced solely from S-adenosylhomocysteine (AdoHcy) through the catalysis of AdoHcy-hydrolase. The direction of AdoHcy-hydrolase activity is determined by its cytosolic substrate concentrations, thereby controlling intracellular AdoHcy levels. Most S-adenosylmethionine (AdoMet)-dependent methyltransferases are regulated in vivo by the ratio of AdoMet/AdoHcy, which is termed “methylation potential” (MP). To test whether high rates of erythropoietin (EPO) expression is reduced by a low MP in vivo we choosed the model of increased EPO production following carbon monoxide (CO) exposure in rats in which high transcriptional activity is responsible for renal EPO production. Results: To induce a sustained hyperhomocysteinemia in rats, we infused i.v. a low or high dose of Hcy resulting in Hcy plasma levels of 87.4±6.2 and 300.8±23.7 µmol/l, respectively. Renal tissue contents of AdoHcy, AdoMet, and adenosine (Ado) were measured after freeze clamp by means of HPLC. Within 4h of CO exposure EPO serum levels increased from 13.6±0.4 (control) to 2254.8±278.3 mIU/ml. Only high dose of Hcy reduces both, the MP from 40.8±2.0 to 8.2±1.0 in the kidney as well as EPO serum levels by 40% compared to control rats. Conclusion: Our data show that severe hyperhomocysteinemia (HHcy) affects the MP in the renal tissue and lowers EPO expression following CO induced intoxication. This result supports the concept that efficient EPO production requires an unimpaired MP.

In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)‐β‐Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin

Mannopeptimycin, a potent drug lead, has superior activity against difficult-to-treat multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). (2S,3S)-beta-Methylphenylalanine is a residue in the cyclic hexapeptide core of mannopeptimycin, but the synthesis of this residue is far from clear. We report here on the reaction order and the stereochemical course of reaction in the formation of (2S,3S)-beta-methylphenylalanine. The reaction is executed by the enzymes MppJ and TyrB, an S-adenosyl methionine (SAM)-dependent methyltransferase and an (S)-aromatic-amino-acid aminotransferase, respectively. Phenylpyruvic acid is methylated by MppJ at its benzylic position at the expense of one equivalent of SAM. The resulting beta-methyl phenylpyruvic acid is then converted to (2S,3S)-beta-methylphenylalanine by TyrB. MppJ was further determined to be regioselective and stereoselective in its catalysis of the formation of (3S)-beta-methylphenylpyruvic acid. The binding constant (K(D)) of MppJ versus SAM is 26 microM. The kinetic constants with respect to k(cat Ppy) and K(M Ppy), and k(cat SAM) and K(M SAM) are 0.8 s(-1) and 2.5 mM, and 8.15 s(-1) and 0.014 mM, respectively. These results suggest SAM has higher binding affinity for MppJ than Ppy, and the C--C bond formation in betamPpy might be the rate-limiting step, as opposed to the C--S bond breakage in SAM.

A fast and efficient method for quantitative measurement of S-adenosyl- l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl- l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis–Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.

Coupling Sequence-specific Recognition to DNA Modification

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is approximately 28 A away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome

BackgroundLeigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or...

Structure of the Thiostrepton Resistance Methyltransferase·S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

Functional Analysis of MycE and MycF, Two O‐Methyltransferases Involved in the Biosynthesis of Mycinamicin Macrolide Antibiotics

Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg(2+). With the reconstituted in vitro assays, the order of mycinamicin VI-->III-->IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers

S′adenosyl-l-methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene, polyamines, biotin, and nicotianamine in plants. Only limited information is available regarding its synthesis (SAM cycle) and its concentrations in plant tissues. The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g−1 fresh weight. Troughs of SAM levels were measured in the evening and night, which corresponds to the time when the major floral scent compound, methyl benzoate, is synthesized by a SAM dependent methyltransferase (NsBSMT) and when this enzyme possesses its highest activity. The SAM synthetase (NsSAMS1) and methionine synthase (NsMS1) are enzymes, among others, which are involved in the synthesis and regeneration of SAM. Respective genes were isolated from a N. suaveolens petal cDNA library. Transcript accumulation patterns of both SAM regenerating enzymes matched perfectly those of the bifunctional NsBSMT; maximum mRNA accumulations of NsMS1 and NsSAMS1 were attained in the evening. Ethylene, which is synthesized from SAM, reached only low levels of 1–2 ppbv in N. suaveolens flowers. It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-009-9490-1) contains supplementary material, which is available to authorized users.

Phosphoanalogues of amino acids involved in methionine metabolism as a new source of antiviral compounds

84 Biological methylation is of primary importance in cell metabolism [1]. The major donor of methyl groups used in biological methylation is S-adenosylmethionine (Met(Ado)), which is converted to S-adenosylhomocysteine Hcy(Ado), the only inhibitor of methylation reactions. The intracellular level of Met(Ado) is regulated by hydrolase (Fig. 1). The nucleoside inhibitors of this enzyme increase the concentration of S-adenosylhomocysteine, which results in suppression of activity of Met(Ado)-dependent methyltransferases, including viral mRNA methylases, and determines the antiviral activity of such compounds [1, 2]. The selectivity of effect of hydrolase inhibitors is low. These compounds inhibit methylation of the main classes of biological molecules, which determines the toxicity of antiviral compounds belonging to the group of nucleoside inhibitors. The search for specific inhibitors of Met(Ado) exchange and methionine metabolism (Fig. 1) remains to be a promising direction in the chemotherapy of viral infections [2].

An intact SAM‐dependent methyltransferase fold is encoded by the human endothelin‐converting enzyme‐2 gene

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment and a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptidemore » with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM-MT and protease domains in a single polypeptide and their transmembrane interplay. Although sequence conservation across the SAM-MT family is weak, the structural fold is highly conserved. The most conserved part of this fold is the SAM-binding subdomain, which is shared between MGC2408 and hypothetical protein TT1324. Typically, the SAM-binding subdomain is flanked by a variable Nterminal extension and, at the C-terminus, by a substrate- binding subdomain, which varies enormously in size but preserves a conserved topology with three antiparallel b-strands. The 'elongated' transcript of ece2 lacks this substrate-binding subdomain. To test the hypothesis that the 255 residue ece2 gene product MGC2408 represents a complete SAM-MT fold, we have determined a crystal structure of this protein in the presence of SAH.« less

Assays for S‐Adenosylmethionine (AdoMet/SAM)‐Dependent Methyltransferases

Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme-coupled continuous spectrophotometric assay used to quantitatively characterize S-adenosyl-L-methionine (AdoMet or SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet-dependent methyltransferase, is hydrolyzed to S-ribohomocysteine and adenine by recombinant AdoHcy nucleosidase. Subsequently, the adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant adenine deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput.

Tapetum‐specific location of a cation‐dependent O‐methyltransferase in Arabidopsis thaliana

Cation- and S-adenosyl-L-methionine (AdoMet)-dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT-like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi-suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N ',N ''-bis-(5-hydroxyferuloyl)-N '''-sinapoylspermidine compensated for by N(1),N(5),N(10)-tris-(5-hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5-hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi-mediated suppression data of the corresponding gene suggest a role of this cation-dependent CCoAOMT-like protein in the stamen/pollen development of A. thaliana.