Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.     

Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus

S-Adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the carboxyl group of benzoic acid to make the volatile ester methyl benzoate, one of the most abundant scent compounds of snapdragon, Antirrhinum majus. The enzyme was purified from upper and lower petal lobes of 5- to 10-day-old snapdragon flowers using DE53 anion exchange, Phenyl-Sepharose 6FF, and Mono-Q chromatography. The purified protein has a pH optimum of 7.5 and is highly specific for benzoic acid, with no activity toward several other naturally occurring substrates such as salicylic acid, cinnamic acid, and their derivatives. The molecular mass values for native and denatured protein were 100 and 49 kDa, respectively, suggesting that the active enzyme is a homodimer. The addition of monovalent cations K+ and NH4+ stimulates BAMT activity by a factor of 2, whereas the addition of Fe2+ and Cu2+ has a strong inhibitory effect. Plant-purified BAMT has Km values of 28 microM and 1.1 mM for SAM and benzoic acid, respectively (87 microM and 1.6 mM, respectively, for plant BAMT expressed in Escherichia coli). Product inhibition studies showed competitive inhibition between SAM and S-adenosyl-L-homocysteine (SAH), with a Ki of 7 microM, and noncompetitive inhibition between benzoic acid and SAH, with a Ki of 14 microM.     

Localization of methyl benzoate synthesis and emission in Stephanotis floribunda and Nicotiana suaveolens flowers

The emission of fragrances can qualitatively and quantitatively differ in different parts of flowers. A detailed analysis was initiated to localize the floral tissues and cells which contribute to scent synthesis in STEPHANOTIS FLORIBUNDA (Asclepiadaceae) and NICOTIANA SUAVEOLENS (Solanaceae). The emission of scent compounds in these species is primarily found in the lobes of the corollas and little/no emission can be attributed to other floral organs or tissues. The rim and centre of the petal lobes of S. FLORIBUNDA contribute equally to scent production since the amount of SAMT (salicylic acid carboxyl methyltransferase) and specific SAMT activity compensate each other in the rim region and centre region. IN SITU immunolocalizations with antibodies against the methyl benzoate and methyl salicylate-synthesizing enzyme indicate that the adaxial epidermis with few subepidermal cell layers of S. FLORIBUNDA is the site of SAMT accumulation. In N. SUAVEOLENS flowers, the petal rim emits twice as much methyl benzoate due to higher total protein concentrations in the rim versus the petal centre; and, both the adaxial and abaxial epidermis house the BSMT (salicylic acid/benzoic acid carboxyl methyltransferase).     

植物SABATH甲基转移酶研究进展

近年来对植物甲基转移酶（methyltransferases,MTs）的研究发现了新一类成员,并用最初发现的3个酶将其命名为SA-BATH甲基转移酶（SABATHMTs）,这3个酶分别是水杨酸羧基甲基转移酶（salicylic acid carboxyl methyltransferases,SAMT）、苯甲酸羧基甲基转移酶（benzoic acid carboxyl methyltransferases,BAMT）和可可碱合酶（theobromine synthase）。SABATHMTs能对植物激素和其他一些小分子物质进行N位或O位甲基化形成相应的甲基化产物,在植物次生代谢、发育及防御中起重要作用。本文从SABATHMTs潜在底物、进化及调控等方面综述了近年来对该家族的研究。     

Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.     

Cellular and subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers.

The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.     

Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic,salicylic, and indole-3-acetic acids in an assay for adenylation

Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.     

An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense

Emission of methylsalicylate (MeSA), and occasionally of methylbenzoate (MeBA), from Arabidopsis thaliana leaves was detected following the application of some forms of both biotic and abiotic stresses to the plant. Maximal emission of MeSA was observed following alamethicin treatment of leaves. A gene (AtBSMT1) encoding a protein with both benzoic acid (BA) and salicylic acid (SA) carboxyl methyltransferase activities was identified using a biochemical genomics approach. Its ortholog (AlBSMT1) in A. lyrata, a close relative of A. thaliana, was also isolated. The AtBSMT1 protein utilizes SA more efficiently than BA, whereas AlBSMT1 catalyzes the methylation of SA less effectively than that of BA. The AtBSMT1 and AlBSMT1 genes showed expression in leaves under normal growth conditions and were more highly expressed in the flowers. In A. thaliana leaves, the expression of AtBSMT1 was induced by alamethicin, Plutella xylostella herbivory, uprooting, physical wounding, and methyl jasmonate. SA was not an effective inducer. Using a beta-glucuronidase (GUS) reporter approach, the promoter activity of AtBSMT1 was localized to the sepals of flowers, and also to leaf trichomes and hydathodes. Upon thrip damage to leaves, AtBSMT1 promoter activity was induced specifically around the lesions.     

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.     

Evolution of Cinnamate/p-coumarate carboxyl methyltransferases and their role in the biosynthesis of methylcinnamate

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.     

Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme

We used a combined evolutionary and experimental approach tobetter understand enzyme functional divergence within the SABATHgene family of methyltransferases (MTs). These enzymes catalyzethe formation of a variety of secondary metabolites in plants,many of which are volatiles that contribute to floral scentand plant defense such as methyl salicylate and methyl jasmonate.A phylogenetic analysis of functionally characterized membersof this family showed that salicylic acid methyltransferase(SAMT) forms a monophyletic lineage of sequences found in severalflowering plants. Most members of this lineage preferentiallymethylate salicylic acid (SA) as compared with the structurallysimilar substrate benzoic acid (BA). To investigate if positiveselection promoted functional divergence of this lineage ofenzymes, we performed a branch-sites test. This test showedstatistically significant support (P < 0.05) for positiveselection in this lineage of MTs (d_N/d_S = 10.8). A high posteriorprobability (pp = 0.99) identified an active site methionineas the only site under positive selection in this lineage. Toinvestigate the potential catalytic effect of this positivelyselected codon, site-directed mutagenesis was used to replaceMet with the alternative amino acid (His) in a Datura wrightiifloral–expressed SAMT sequence. Heterologous expressionof wild-type and mutant D. wrightii SAMT in Escherichia colishowed that both enzymes could convert SA to methyl salicylateand BA to methyl benzoate. However, competitive feeding withequimolar amounts of SA and BA showed that the presence of Metin the active site of wild-type SAMT resulted in a >10-foldhigher amount of methyl salicylate produced relative to methylbenzoate. The Met156His-mutant exhibited little differentialpreference for the 2 substrates because nearly equal amountsof methyl salicylate and methyl benzoate were produced. Evolutionof the ability to discriminate between the 2 substrates by SAMTmay be advantageous for efficient production of methyl salicylate,which is important for pollinator attraction as well as pathogenand herbivore defense. Because BA is a likely precursor forthe biosynthesis of SA, SAMT might increase methyl salicylatelevels directly by preferential methylation and indirectly byleaving more BA to be converted into SA.