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First published online August 13, 2004; 10.1104/pp.104.041806 Plant Physiology 135:1946-1955 (2004) © 2004 American Society of Plant Biologists Biochemical and Structural Characterization of Benzenoid Carboxyl Methyltransferases Involved in Floral Scent Production in Stephanotis floribunda and Nicotiana suaveolens1Department of Biological Sciences, University of Rostock, 18059 Rostock, Germany (M.B.P., F.H., S.S., U.E., B.P.); Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (F.C., E.P.); Department of Plant Physiology Bio III, Rheinisch-Westfälische Technische Hochschule Aachen University, 52074 Aachen, Germany (I.K., A.S.); and Structural Biology Laboratory, The Salk Institute for Biological Studies and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, 92307 (J.R., J.P.N.)
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 Km 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 Km 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.
Methyl salicylate and methyl benzoate are common components of floral scent and are believed to be important attractants of insect pollinators (Dobson, 1994  ). Enzymes that catalyze the formation of methyl salicylate and methyl benzoate from salicylic acid (SA) and benzoic acid (BA), respectively, have been reported from flowers of Clarkia breweri, snapdragon (Antirrhinum majus), petunia (Petunia hybrida), and Stephanotis floribunda (Ross et al., 1999; Murfitt et al., 2000; Negre et al., 2002; Pott et al., 2002). While these enzymes use S-adenosyl-L-met (SAM) as the methyl donor as do many previously characterized methyltransferases that act on a variety of substrates (e.g. DNA, protein, phenylpropanoids), these SA and BA carboxyl methyltransferases (SAMT and BAMT, respectively) display primary amino acid sequences that show no significant sequence identity to other methyltransferases. Interestingly, it was subsequently discovered that a group of N-methyltransferases involved in the biosynthesis of the alkaloid caffeine, including theobromine synthase, also share sequence similarity with SAMT and BAMT (D'Auria et al., 2003). These enzymes were therefore grouped into a new class of methyltransferases designated the SABATH methyltransferases, and this family now also includes jasmonic acid methyltransferase (Seo et al., 2001) and indole-acetic acid methyltransferase (Zubieta et al., 2003). The recently obtained three-dimensional structure of C. breweri SAMT (Zubieta et al., 2003) also clearly indicates that these enzymes have a unique structure distinct from those of unrelated methyltransferases found in plants (Noel et al., 2003).
It is estimated that the total number of different compounds with specialized functions synthesized by various plant species exceeds 100,000 but that each species synthesizes only a small fraction of the potential chemical diversity of the plant kingdom (Pichersky and Gang, 2000
The floral enzymes involved in benzenoid ester production provide an excellent opportunity to examine the issue of enzyme specificity and promiscuity in specialized metabolism and the role of each in the evolution of plant-specialized metabolism. BA and SA are very similar compounds, differing only in the presence of an ortho hydroxyl group on the benzyl ring of SA (Fig. 1), yet some flowers synthesize one or the other of these two methyl ester derivatives and other species synthesize both. For example, S. floribunda flowers emit primarily methyl benzoate and little methyl salicylate (Pott et al., 2002
Emission of Methyl Salicylate and Methyl Benzoate from Flowers of N. suaveolens and S. floribunda
While the emission of S. floribunda flowers has been characterized (Matile and Altenburger, 1988
Carboxyl Methyltransferase Activities in Petals of N. suaveolens and S. floribunda To determine whether carboxyl methylation activities are present in these flowers, protein extracts from petals were incubated with BA, SA, and several other related compounds as well as with jasmonic acid (Fig. 1) together with [14C]SAM. The extract of S. floribunda showed the highest activity with SA as well as relatively high levels of activity with several related hydroxylated BA derivatives (Table II). The N. suaveolens extract possessed the highest activity with 2-methoxy BA (o-anisic acid) and 4-hydroxy BA (Table II). Little methylation activity was observed with jasmonic acid. S. floribunda flowers emit about 15-fold less methyl salicylate than methyl benzoate (Table I), which is a contradictory reflection of the 9-fold higher SA-methylating activitiy in the flower extract of S. floribunda. In N. suaveolens, which also emits more methyl benzoate than methyl salicylate (4-fold), BA-methylating activity was 4-fold higher than SA-methylating activity. Moreover, the overall methylating activity levels in S. floribunda per g FW were much higher than the methylating activity levels found in the floral extract of N. suaveolens (Table II).
Isolation of cDNAs Encoding Floral Benzenoid Carboxyl Methyltransferases from S. floribunda and N. suaveolens, and Biochemical Characterization of the Encoded Enzymes
The isolation of a cDNA from S. floribunda that encodes an enzyme capable of methylating SA was previously reported (Pott et al., 2002
The two cDNAs from S. floribunda and N. suaveolens were cloned into the pET101 bacterial expression vector, expressed in Escherichia coli, and purified Fig. 3. The purified enzymes were assayed with a range of substrates at a concentration that should saturate or nearly saturate the enzyme (Table II). The S. floribunda enzyme was most active with SA, while the N. suaveolens protein was most active with BA. However, each protein also methylated a broad range of benzoic and cinnamic acid derivatives with relative activities between 1% and 99% compared to their preferred substrates (Table II). It appears that the spectrum of possible substrates (greater than 20% relative activity) is broader for the S. floribunda enzyme than for the enzyme from N. suaveolens.
The Km values of the enzymes for some of these substrates were determined (Table III). The S. floribunda enzyme displayed a Km value for SA that was almost 12-fold lower than its Km value for BA. 3-Hydroxy BA was preferred over BA; the Km value for 3-hydroxy BA was 7-fold lower than the Km value for BA. The turnover number (Kcat) of the enzyme with BA was 10-fold higher than with SA; however, the overall catalytic efficiency (Kcat/Km) of the enzyme with SA was only slightly higher than with BA (Table III). The Km values of the N. suaveolens enzyme for SAs and BAs were similar (Table III), but the turnover number with BA was 3-fold higher than with SA (Table III). A 4-fold higher catalytic efficiency with BA over SA was determined for the N. suaveolens BSMT.
Determination of Endogenous Concentrations of SA and BA
For scent enzymes that can use more than one substrate, it is conceivable that the amount of synthesized and emitted esters depends at least in part on the availability of specific substrates (Kolosova et al., 2001
Molecular Models of the Benzenoid Carboxyl Methyltransferases
A detailed study of the crystal structure of SAMT from C. breweri, including the identification of active site residues, was recently reported (Zubieta et al., 2003
The substitutions in the S. floribunda enzyme allow for the accommodation of bulkier substrates, and so do those in the N. suaveolens enzyme, but to a lesser extent. The modeling results are consistent with the empirical observation that the S. floribunda enzyme exhibits the broadest substrate tolerance during methylation, accepting 11 of the 17 tested substrates with 20% to 100% relative activity (Table II). The S. floribunda enzyme has the lowest Km value with SA (and is therefore designated SAMT), likely because this acceptor substrate's 2-hydroxyl position forms an intramolecular hydrogen bond with the substrate's own carboxyl group, thus stabilizing the substrate in an orientation favorable for methylation (Zubieta et al., 2003 ). A substrate with a hydroxyl at the meta position is less favored by S. floribunda SAMT, but would permit the formation of a hydrogen bond to Tyr-261 that would provide some stability upon substrate binding. A 4-hydroxyl group does not lie within hydrogen bonding distance of protein residues in the rendered models, so it would appear not to provide additional stabilizing contacts with the active site pocket. However, the fact that the enzyme accepts 3,4-hydroxy BA no better than 4-hydroxy BA suggests that the 4 position substitution may sterically hinder substrate binding perhaps due to clashes with the nearby residues Phe-355, Leu-232, or Val-316. This enzyme disfavors substrates with substitutions at positions 5 and 6, which, again, may prevent proper substrate positioning due to steric conflicts with surrounding residues identified in the models and including Phe-355, Gln-25, Tyr-153, or Val-316. Because this enzyme possesses a relatively expansive active site, it can also accommodate bulkier substrates such as cinnamic acid and caffeic acid. The N. suaveolens enzyme exhibits higher catalytic rates at saturating substrate concentrations for BA and 4-hydroxy BA, but has similar Km values for BA and SA (Tables II and III) and is therefore designated BSMT. 4-Hydroxy BA could potentially be stabilized in the enzyme active site by hydrogen-bonding interactions with Ser-344 or Tyr-263, while Phe-307 forms a clamp on the substrate that may prevent optimal substrate positioning whenever there is a substitution at the meta position on the benzoic ring. In the case of 2-methoxy BA, the 2-methoxy group of the substrate likely hydrogen bonds with the ring nitrogen of His-158, thus constraining the substrate in a more favorable position for methylation. This enzyme also displays poor activity with substrates possessing substitutions at positions 5 or 6, likely due to spatial conflicts with nearby residues Phe-24 (not shown), Met-218, Gln-25, or Phe-155 and, unlike S. floribunda SAMT, cannot accommodate bulkier substrates.
Flower-specific expression of S. floribunda SAMT was previously reported (Pott et al., 2002
Contribution of S. floribunda SAMT and N. suaveolens BSMT to the Synthesis of Benzenoid Methylesters Emitted from Their Flowers The N. suaveolens emits more methyl benzoate than methyl salicylate, which is in agreement with the flower-specific enzyme we have characterized here that can methylate BA more efficiently than SA. Southern-blot analysis does not indicate the presence of additional floral benzenoid carboxyl homologous sequences (data not shown). It is therefore reasonable to conclude that this N. suaveolens BSMT is responsible for the synthesis of both methyl benzoate and methyl salicylate in a physiological setting, and that the amount of each methylated benzenoid produced is largely determined by the internal availability of the respective substrates, with BA levels far exceeding SA levels in plant tissue (Table IV). A different situation appears to occur in the flowers of S. floribunda. We have identified a single flower-specific benzenoid carboxyl methyltransferase enzyme from S. floribunda that has similar catalytic efficiency with SA and BA, although the Km value for SA is 10-fold lower. S. floribunda petal extracts also exhibit higher methylation activity with SA than with BA (Table II). Yet, these flowers emit 15-fold more methyl benzoate than methyl salicylate (Table I). This latter observation is most consistent with the additional observation that the concentration of BA in the floral tissue is 1,000- to 2,000-fold higher than the concentration of SA (Table IV).
Biochemical characterization and structural elucidation of the first benzenoid carboxyl methyltransferase identified, SAMT from C. breweri, indicate that this enzyme is relatively specific for SA, although it does methylate BA and a few other similar BA derivatives but at a much lower efficiency then the characterized physiological substrate, SA (Ross et al., 1999 The modeling of the active sites of flower-specific S. floribunda SAMT and N. suaveolens BSMT shows that these enzymes are able to accommodate several substrate derivatives as well as more bulky substrates, and are therefore less specific than the SAMT of C. breweri. However, kinetic measurements show that while the S. floribunda enzyme possesses a lower Km value for SA, its catalytic efficiency is similar with BA and SA, and the N. suaveolens enzyme has similar Km values with SA and BA but is almost 4-fold more efficient with BA (Table III). These observations are explained in part by steric considerations in the active site (Fig. 4, A and B). However, clear differences in efficiency with specific substrates are not, in general, due entirely to factors residing in the active site. Such a situation is demonstrated by comparison of the active sites of AtBSMT and AlBSMT which are identical (Fig. 2A; Fig. 4, C and D), while their efficiencies for BA and SA are reversed, presumably due to differences elsewhere in the protein that affect the structure, dynamics, or both in the enzyme in solution.
It has been argued that broad substrate promiscuity and concomitant low turnover rates are traits of newly evolved enzymes of secondary metabolism and that over time such enzymes may evolve a restricted substrate range and a faster turnover rate because such changes increase fitness (Firn and Jones, 2000
Whether the broad specificity of the two benzenoid carboxyl methyltransferases, whose structural and biochemical characterization is reported here, is due to recent adaptative changes or due to long-term adaptation is still unclear. Addressing this question will require additional investigations into the involvement of these enzymes in the production of a range of methyl esters that S. floribunda and N. suaveolens flowers synthesize under different environmental conditions and will necessitate a larger scale comparison of these volatile chemical mixtures and the underlying genetic fingerprint across related species existing in unique ecotypes. This comparative study would require a detailed and multifaceted approach using orthologous genes and enzymes in related species but referenced to specific environments wherever possible so as to deconvolute the selective pressure exhibited by the local environment over the natural genetic drift that may occur over time. It is interesting to note that H. carnosa, a close relative of S. floribunda, which emits methyl salicylate but not methyl benzoate from its flowers (Altenburger and Matile, 1988
Plant Material and Plant Growth Conditions Stephanotis floribunda (Brongn.) plants were grown in the greenhouse with supplemental light between 6 AM and 10 PM during autumn and winter. Nicotiana suaveolens (Lehmann) was grown on vermiculite and watered with Hoagland solution. The plants were kept in growth rooms at 18°C to 22°C with 16 h light (6 AM till 10 PM) of 150 µmol m–2 s–1. Day 1 was the day of flower opening.
The crude extracts were prepared as described in Wang et al. (1997)
The enzyme assay was performed according to Wang et al. (1997) For product verification the same assay was scaled up to 1 mL with nonradioactive SAM. The products were extracted with 1 mL of hexane and analyzed via gas chromatography-mass spectrometry on a DB-5 column (60 m x 0.25 mm (injector 200°C, interface 235°C, oven program starting at 50°C, hold 2 min, heating to 275°C at a rate of 15°C/min, hold 10 min). Products were identified via mass spectrometry profile and methyl salicylate, methyl benzoate, and methyl jasmonate could also be identified via retention time and standard chemicals. In all kinetics studies (e.g. Km value), appropriate enzyme concentration and incubation times were chosen so that the reaction velocity was linear during the incubation time period; at least three replicates were performed. To measure the Km for each substrate, one substrate concentration was fixed at a saturated level (usually 1 mM; 10 mM BA for Km determination of S. floribunda SAMT) and the concentration of the other substrate to be measured was varied. Lineweaver-Burk, Hanes, and Eadie-Hofstee plots were performed to obtain the Km and Kcat values.
RT-PCR
5'-RACE and 3'-RACE For the 3'-RACE, total RNA from flowers harvested at midnight on the day of anthesis was used. To eliminate contaminating DNA, a digest with 1 unit DNaseI (Sigma-Aldrich) was performed according to the manufacturer's recommendations. Ten microliters (1.3 µg) RNA from this reaction was used for RT-PCR with the eAMV kit for RT-PCR (Sigma-Aldrich). Three different primers were used in separate reactions: oligo-d(T)15A, oligo-d(T)15C, and oligo-d(T)15G, linked to an adaptor sequence for the RT reaction (5'-GACTGGACTTCAATCAGTTAC(TTT)5A/C/G-3', respectively). For the RT reaction the following temperature program was adjusted: 15 min 45°C, temperature increment of 2°C every 3 min up to 55°C, hold 15 min, temperature increment of 2°C every 3 min up to 65°C, hold 15 min. Five microliters were then used for the following PCR with a gene-specific and an adaptor primer (5'-GAAGATCGCTTTAGCAAAGCT-3' and 5'-GACTGGACTTCAATCAGTTAC-3', respectively), according to the manufacturer's manual. Gel electrophoresis revealed a fragment of about 450 bp, which was recovered from the agarose gel using a Gel Extraction kit (Qiagen). Cloning and sequencing was performed as described in the previous paragraph. The accession number of the N. suaveolens BSMT is AJ628349.
The full-length N. suaveolens BSMT cDNA obtained by RT-PCR and the additional fragment obtained by the 3'-RACE were linked via a SacI restriction site and the resulting fragment was amplified with Pfu DNA polymerase (Promega) and cloned into the expression vector using the pET101 Directional TOPO Expression kit according to the manufacturer's instructions. Overexpression of the poly-His (6x his) tagged gene was performed in the Escherichia coli strain HMS174 (F–, recA, [r– K12, m+ K12], Rifr) BL21 (DE3) pLys. The DNA of the complete S. floribunda SAMT gene (including start-to-stop codons) was amplified by PCR (primers STSAMT forward and reverse) and cloned without a poly-His tag using the pCR T7/CT TOPO TA Cloning kit (Invitrogen, Karlsruhe, Germany) and transformed into E. coli BL21 (codon+) cells. Overexpressed protein was obtained after 2 h of preincubation at 37°C, induction with 1 mM isopropylthio-
At indicated time points, flower petals (300–1,000 mg) were harvested, frozen in liquid nitrogen, and RNA was extracted according to Cheng and Seemann (1998) Hybridization was performed with 5x SSC buffer (7% SDS, 50% formamide, 0.1% lauryl sarcosine, 2% blocking solution, 50 mM Na2HPO4) at 50°C overnight. Membranes were washed twice for 5 min with 2x SSC, 0.1% SDS at room temperature and for 15 min with 0.1x SSC and 0.1% SDS at 50°C. After hybridization, the membranes were incubated with anti-Dig alkaline phosphate and CSPD (disodium 3-(4-methoxyspiro[lsqb]1,2-dioxetane-3,2[prime]-{5[prime]-chloro}tricyclo{3.3.1.13,7}decan[rsqb]4-yl; Roche Diagnostics) was used as a substrate. The chemiluminescence signal detected and quantitated with the LAS-1000 (Fuji-Raytest; Straubenhardt, Germany; software Image Gauge, Fujifilm, Tokyo) for 10 to 60 min. Transcript levels were normalized to rRNA levels, which were determined by repeated hybridizations, and the BSMT mRNA/rRNA ratios were calculated. The highest ratio of each individual blot was set to 100% and SE was calculated.
The extraction protocol was as described by Meuwly and Metraux (1993)
Modeling of the active sites of the N. suaveolens BSMT, the S. floribunda SAMT, and the Arabidopsis BSMT enzymes was accomplished by fitting the amino acid sequences of each of the three proteins to the previously published three-dimensional structure of the C. breweri SAMT (Zubieta et al., 2003 Sequence data (for the N. suaveolens BSMT) from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ628349 (for the N. suaveolens BSMT).
We thank Claudia Dinse for technical assistance, and Claus Schnarrenberger and Joachim Schroeder for initial helpful discussions. Received February 27, 2004; returned for revision May 26, 2004; accepted May 26, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft Pi 153/17–1 and 17–2 to B.P.), by Cusanuswerk (to M.B.P.), and by the National Science Foundation (grant nos. IBN–0211697 and MCB–0312449 to E.P. and MCB–0312466 to J.P.N.). 
2 Present address: Carnegie Institution of Washington, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305.
3 Present address: Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041806. * Corresponding author; e-mail birgit.piechulla{at}biologie.uni-rostock.de; fax 49–(0)381–4986132.
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Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, Noel JP (2003) Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell 15: 1704–1716 This article has been cited by other articles:
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ABSTRACT
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-galactoside and continued incubation for 20 h at 20°C. The cells were harvested by centrifugation at 6,000g for 15 min, resuspended in 5 mL of lysis buffer (10 mM NaCl; 50 mM Tris/HCl, pH 8.0; 1 mM EDTA; 10% [w/v] glycerol; 14.3 mM mercaptoethanol), sonicated three times for 30 s, and the soluble extract centrifuged at 21,000g. Extract activity assays with 10 µL of protein fractions were performed as described above and proteins were separated on 12.5% SDS polyacrylamide gels. 
