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Archives of Biochemistry and Biophysics Vol. 382, No. 1, October 1, pp. 145–151, 2000 doi:10.1006/abbi.2000.2008, available online at http://www.idealibrary.com onPurification 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 1Lisa M. Murtt,* ,2 Natalia Kolosova,* ,2 Craig J. Mann, and Natalia Dudareva* ,3*Department of Horticulture and Landscape Architecture and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907Received June 7, 2000S-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 puried from upper and lower petal lobes of 5- to 10-day-old snapdragon owers using DE53 anion exchange, Phenyl-Sepharose 6FF, and Mono-Q chromatography. The puried protein has a pH optimum of 7.5 and is highly specic 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 NH 4 stimulates BAMT activity by a factor of 2, whereas the addition of Fe 2 and Cu 2 has a strong inhibitory effect. Plant-puried BAMT has K m values of 28 M and 1.1 mM for SAM and benzoic acid, respectively (87 M 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 K i of 7 M, and noncompetitive inhibition between benzoic acid and SAH, with a K i of 14 M. 2000 Academic Press1 This work is supported by National Science Foundation Grant IBN-9904910 and by grants from the Fred Gloeckner Foundation, Inc. This paper is Contribution 16268 from the Purdue University Agricultural Experimental Station. 2 These authors made equal contributions to this work. 3 To whom correspondence should be addressed. Fax: (765) 4940391. E-mail: dudareva@hort.purdue.edu.Key Words: oral scent; methyl benzoate; O-methyltransferase; snapdragon; avor; benzoic acid.Methyl esters such as methyl benzoate, methyl cinnamate, methyl jasmonate, and methyl salicylate are common scent components in many plant species that contribute signicantly to the total oral scent output and play an important role as attractants for pollinators (1–3). In addition, such volatiles may function as airborne signals that activate disease resistance via the expression of defense-related genes in neighboring plants and in the healthy tissues of infected plants (4 – 6) or in tritrophic interactions by attracting natural enemies of the herbivores upon herbivore damage (7). Although many plant S-adenosyl-L-methionine (SAM) 4 dependent methyltransferases can be found in sequence databases, to date, none of these genes, with the exception of Clarkia breweri SAM:salicylic acid carboxyl methyltransferase (SAMT) (8), is known to be involved in the formation of volatile methyl esters (9). Methyl benzoate has been reported in the oral scent of more than 30 different species (1); however, no plant enzyme catalyzing its formation has been puried and characterized. We have recently isolated a cDNA encoding S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT), the nal enzyme in the biosynthesis of4 Abbreviations used: BA, benzoic acid; BAMT, S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase; SAM, S-adenosylL-methionine; SAMT, S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase.0003-9861/00 $35.00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved.145

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146MURFITT ET AL. range were pooled (total of 26 mL) and loaded on a Phenyl-Sepharose 6 Fast Flow (low substitution; level of phenyl substitution is 20 mol per milliliter of gel) column (0.7 2.5 cm, Pharmacia Biotech Inc.) attached to a Pharmacia FPLC apparatus and preequilibrated with 300 mM KCl in Buffer A at a ow rate of 0.2 mL/min. After the enzyme was loaded, the column was washed with 3 mL of 300 mM KCl in Buffer A and eluted with a linear reverse gradient (4 mL) from 300 mM KCl in Buffer A to water followed by an additional 11 mL of water. Fractions of 0.7 mL were collected into tubes containing 100 L of 1 M KCl, 100 L of glycerol, 100 L of 0.5 M Bis–Tris–HCl, pH 6.9, and 1 L of 14 M -mercaptoethanol. The fractions containing BAMT activity, eluted with water, were pooled (7 mL) and subjected to ion-exchange chromatography on a Mono-Q column using the FPLC system. The column was previously equilibrated with 100 mM KCl in Buffer A. After the protein was loaded onto the column, the column was washed with 100 mM KCl in Buffer A and the bound protein was eluted using a 10-mL linear (100 – 400 mM) gradient of KCl in Buffer A followed by an additional 5 mL of Buffer A containing 400 mM KCl at ow rate of 0.25 mL/min. The enzyme consistently eluted at about 360 mM KCl. Fractions (0.5 mL each) were collected and protein content and purity were examined by SDS–PAGE followed by staining of the gel with Coomassie brilliant blue. Fractions which had the largest amount of pure protein were used for N-terminal sequencing, internal peptide sequencing, and initial enzyme characterization. Expression of BAMT in E. coli and protein purication. The coding region of BAMT was amplied with sense 29-mer oligonucleotide 5 -GTCTAGACATATGAAAGTGATGAAGAAAC-3 (for the rst methionine codon) or 27-mer oligonucleotide 5 -CTCTAGACATATGAAGAAACTTTTGTG-3 (for the second methionine codon) that introduced an NdeI site at the initiating ATG codon and the antisense 29-mer oligonucleotide 5 -TGGATCCTTCATCTCCTACTTAGAGAAAC-3 that introduced a BamHI site downstream of the stop codon. The PCR-amplied 1.1-kb fragment was cloned into the NdeI– BamHI site of the expression vector pET-28a, which contains an N-terminal polyhistidine (6 His) tag (Novagen). E. coli BL21(DE3) cells were transformed with recombinant plasmid and were grown in LB medium with 50 g/mL kanamycin at 37°C. When the culture density reached an OD 600 of 0.5, the expression of BAMT cDNA was induced by addition of IPTG to nal concentration of 0.4 mM. After 20-h incubation with shaking (200 rpm) at 20°C, E. coli cells were harvested by centrifugation and sonicated in lysis buffer, containing 10 mM NaCl, 50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 10% glycerol, and 10 mM -mercaptoethanol. BAMT activity was measured in soluble and insoluble fractions. The E. coli expressed BAMT protein was puried by nickel-based afnity chromatography (2.5-ml bed volume) according to the manufacturer’s protocol (Novagen). Protein was eluted with 10 ml of stripping buffer (0.5 M NaCl, 20 mM Tris–HCl, pH 7.9, and 100 mM EDTA), and the fractions containing BAMT activity were pooled and dialyzed against 2 L of Buffer A overnight at 4°C. Molecular weight determination. Molecular weight of the native plant BAMT protein was determined by gel ltration on a Superdex 200-HR (Pharmacia Biotech) column (1 30 cm) calibrated with the following markers: cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), aldehyde dehydrogenase 3 (100 kDa), alcohol dehydrogenase (150 kDa), and -amylase (200 kDa). Buffer, containing 100 mM Na phosphate, pH 7.4, 100 mM NaCl, and 0.025% -mercaptoethanol, was used for column equilibration and elution. Fractions of 0.2 mL were collected at a ow rate of 0.5 mL/min and analyzed for BAMT activity. Denaturing SDS–PAGE was performed on 10% gels to determine the subunit molecular weight. The gels were calibrated with molecular weight standards in the range 7.4 –208 kDa (Bio-Rad). Temperature effect on BAMT stability. Puried BAMT proteins were incubated at temperatures ranging from 4 to 65°C for 30 min and then chilled on ice. Samples incubated at each temperature weremethyl benzoate, one of the major components of snapdragon oral scent (10). This protein contains 364 amino acids and has strict substrate specicity for benzoic acid. Its sequence does not share any sequence similarity to previously characterized proteins, including O-methyltransferases, with the exception of SAMT from C. breweri, to which it is 40% identical (8). Both enzymes have the ability to transfer the methyl group of SAM to a free carboxyl group and they dene a new class of plant carboxyl methyltransferases (8, 10). Here, we report the purication and characterization of BAMT from snapdragon owers and from Escherichia coli cells expressing snapdragon BAMT cDNA.MATERIALS AND METHODSPlant material. Maryland True Pink snapdragon cultivar (Antirrhinum majus) (Ball Seed Co.) was grown under standard greenhouse conditions, as previously described (10). Upper and lower petal lobes of 5- to 10-day-old owers were used for enzyme isolation as a oral tissue with the highest BAMT specic activity (10). Enzyme extraction. All extraction and purication procedures were carried out at 4°C except as noted. Freshly excised upper and lower petal lobes of snapdragon owers were frozen in liquid N 2 and ground to a ne powder using a mortar and pestle. The frozen powder was immediately slurried with extraction buffer (5:1 (v/w) buffer:tissue) containing 50 mM Bis–Tris–HCl, pH 6.9, 10 mM -mercaptoethanol, 5 mM Na 2S 2O 5, 1% (w/v) polyvinylpyrrolidone (PVP-40), 1 mM phenylmethanesulfonyl uoride, and 10% (v/v) glycerol. The slurry was additionally homogenized in a chilled glass homogenizer (Wheaton, VWR Scientic Products), passed through two layers of Miracloth (Calbiochem), and centrifuged for 10 min at 12,000g. The pellet was discarded and the supernatant that contained the BAMT activity was used as the enzyme source. BAMT enzyme activity. To monitor the BAMT elution prole during purication, enzyme assays were performed as previously described by Dudareva et al. (10). Enzyme activity was determined by measuring transfer of the 14C-labeled methyl group of SAM to the carboxyl group of benzoic acid. The standard reaction mixture (100 L) consisted of 20 L of crude extract (25– 40 g of protein) and 100 M S-adenosyl-L-methionine (SAM; containing 0.1 Ci of S-[methyl14 C]adenosyl-L-methionine (NEN Life Science Products, Boston, MA)) in assay buffer (50 mM Tris–HCl, pH 7.5, and 3 mM -mercaptoethanol) containing 2 mM benzoic acid and 0.5 mM EDTA. After incubation for 30 min at 20°C, the radioactively labeled methylated product was extracted by the addition of 100 L of hexane, and 50 L of the organic phase was counted in a liquid scintillation counter (Model LS6800, Beckman, Fullerton, CA). In assays for pH optimum, cofactor requirements, and K m measurements, proper concentrations of puried BAMT were chosen so that the reaction velocity was proportional to enzyme concentration and was linear with respect to time for at least 30 min. Protein concentration was determined by the Bradford method (11), using the Bio-Rad protein reagent and bovine serum albumin as a standard. Protein purication. In a typical purication procedure, 125 mL of crude extract (representing 25 g of fresh weight of petal tissue) was loaded onto a DEAE– cellulose column (10 mL of DE53, Whatman) preequilibrated with a solution containing 50 mM Bis–Tris– HCl, pH 6.9, 10% glycerol, and 10 mM -mercaptoethanol (Buffer A) at a ow rate of about 1 mL/min. After the unabsorbed material was washed from the column with 30 mL of Buffer A, BAMT was eluted with a linear gradient (60 mL) from 0 to 400 mM KCl in Buffer A. Fractions (2 mL) were collected and assayed for BAMT activity. Fractions with the highest BAMT activity in the 180 –280 mM KCl

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BENZOIC ACID CARBOXYL METHYLTRANSFERASE then used for enzyme assays. At least three independent assays were performed for each point and then an average was taken. pH optimum of BAMT activity. The optimum pH for BAMT activity was determined using two buffer systems. Reactions were carried out in 50 mM Tris–HCl buffer with pH ranging from 6.5 to 9.0 and in 50 mM Tris–Na phosphate–Na citrate buffer with pH ranging from 4.0 to 9.5. Final results are an average of four independent assays. Effectors. Enzyme assays were performed with one of the following cations present in the assay buffer at the nal concentration of 5 mM: Ca 2 , Cu 2 , Fe 2 , K , Mg 2 , Mn 2 , Na , NH 4 , and Zn 2 . Except for Cu 2 and Fe 2 , which precipitate under reducing conditions, all assay buffers also contained 10 mM -mercaptoethanol. Final results are an average of three independent assays. Determination of kinetic properties. Alternative substrate competition experiments were performed by varying the concentration of one substrate at each of a series of concentrations of the other. Data were presented as double-reciprocal plots of initial velocity (v) versus varying substrate (S) concentrations. In all experiments, an appropriate enzyme concentration was chosen so that the reaction velocity was linear during the incubation time period. Substrate interaction studies were done by xing the concentration of one substrate while changing that of the other. Linear regressions were tted to the data in double-reciprocal plots. Replots of the data were used to determine the kinetic parameters.147RESULTS AND DISCUSSIONPurication of BAMT from snapdragon owers. We have previously shown that 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. (10). We have also isolated a snapdragon cDNA clone encoding BAMT and shown that the BAMT gene is only expressed in the upper and lower lobes of petals of snapdragon owers and the levels of BAMT mRNA are positively correlated with BAMT activity. For isolation of a BAMT cDNA, BAMT protein was rst puried from snapdragon owers and subjected to partial peptide sequencing. However, the purication protocol has not been reported, nor the enzyme characterization with respect to its kinetic properties and other parameters (10). In this study, we present detailed protocols for purication of BAMT protein from the crude extract of snapdragon petals and from E. coli cells expressing the Antirrhinum BAMT cDNA and the characterization of the enzyme. Plant BAMT protein was puried from 5to 10-day-old upper and lower petal lobes (oral tissue with the highest BAMT specic activity (10)) using DE53 anion exchange, Phenyl-Sepharose 6FF (low substitution), and Mono-Q chromatography (Fig. 1). The key step in the purication procedure was hydrophobic interaction chromatography using PhenylSepharose 6FF (low substitution), which removed most of the contaminating proteins and yielded a 31-fold purication with recovery of about 8% (Table I and Fig. 2). Further purication was achieved by Mono-Q chro-FIG. 1. Purication of BAMT from petal tissue of snapdragon owers. The purication involved three column chromatographic steps: A, DE53; B, Phenyl-Sepharose 6FF (low substitution); and C, Mono-Q. The dotted lines are amount of protein (A, B) or absorbance at 280 nm (C), and the solid circles represent the BAMT activity expressed relative to the most active fraction (100%) in each chromatographic step. The dashed lines show salt gradients used during purication steps.matography, which resolved one peak of BAMT activity almost free of contaminants when visualized on SDS– PAGE (Fig. 2). This purication protocol resulted in a 112-fold increase in specic activity over the crude extract, with a recovery of 2.2% (Table I). Starting with 25 g of fresh petal material (256 mg of protein), we were able to obtain 53 g of puried BAMT protein with a specic activity 327 pkat/mg of protein. We have previously shown that this protein has strict substrate specicity for benzoic acid and no activity with several other naturally occurring substrates such as salicylic acid, trans-cinnamic acid, and their derivatives (3-hydroxybenzoic acid, 4-hydroxybenzoic acid, benzyl alcohol, and 2-coumaric, 3-coumaric, and 4-coumaric acids) (10). After purication, BAMT protein was subjected to extensive protein sequencing and the amino acid se-

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148MURFITT ET AL. TABLE IBAMT Purication from Antirrhinum majus PetalsTotal protein (mg) 256 a 40 0.68 0.05 Total activity (pkat) 749.6 503.5 62.0 16.4 Specic activity (pkat/mg of protein) 2.9 12.6 91.1 327.5 Purication (fold) 1.0 4.3 31 112 Recovery (%) 100 67.2 8.3 2.2Purication step Crude extract DE53 Phenyl-Sepharose Mono-QaRepresenting 25 g of fresh weight of upper and lower petal lobes of snapdragon owers.quences from six internal regions were determined and matched the sequence encoded by a BAMT cDNA (10). Expression of BAMT cDNA in E. coli also conrmed that the isolated protein is benzoic acid carboxyl methyltransferase, since bacterial cells not only produced enzymatically active protein but also synthesized a small amount of methyl benzoate and secreted it into the medium (10). The molecular mass value of native plant BAMT protein was determined to be 100 kDa from gel ltration chromatography on Superdex 200-HR (Fig. 3), whereas on the SDS–PAGE gels, the denatured enzyme exhibited a single band corresponding to a molecular mass of 49 kDa (Fig. 2). These results suggest that the enzymatically active enzyme exists as a homodimer. The calculated molecular mass of the protein encoded by BAMT cDNA is 41 kDa, which is smaller than the apparent molecular mass calculated from its migration in SDS–PAGE gels. Such discrepancies are not uncommon (12–14), and in this case may be caused, at least in part, by a relatively high percentage (13.9%) of negatively charged residues in the middle part of the protein and two positively charged clusters at the amino and carboxyl ends of the protein.pH optimum and ion requirements. The pH dependence of BAMT activity was examined in the pH range 4.0 –9.5 using snapdragon BAMT puried both from snapdragon petal tissue and from E. coli (starting from the rst and second methionine codons; see below). The plant-puried BAMT was found to be very similar to the E. coli expressed snapdragon BAMT. The pH optimum for the BAMT protein was 7.5, with 65% of maximum activity at both pH 6.5 and 8.5. At pH 5.5 and 9.5, the enzyme activity fell to about 50% of the optimal value. The enzyme was active in both Tris- and phosphate– citrate-based buffers. BAMT activity (again, puried both from petals and from E. coli) was not affected by the presence of 5 mM Mg 2 in the assay reaction. The addition of monovalent cations K and NH 4 stimulated BAMT activity by a factor of 2, whereas the addition of Fe 2 and Cu 2 has a strong inhibitory effect (75–100% inhibition). Other cations such as Zn 2 , Na , Ca 2 , and Mn 2 affect BAMT activity only slightly ( 10%).FIG. 2. SDS–PAGE analysis of purication stages for BAMT. Active fractions from each purication step were separated by 13% SDS–PAGE and stained with Coomassie brilliant blue. Position of molecular weight markers is indicated on the left. Lane 1, crude extract ( 40 g); lane 2, DE53 ( 30 g); lane 3, Phenyl-Sepharose ( 5 g); lane 4, Mono-Q ( 5 g); lane 5, E. coli expressed BAMT protein (Met 1) after purication on nickel column ( 3 g).FIG. 3. Native gel ltration chromatography of snapdragon plant puried BAMT. The Mono-Q puried enzyme was separated on a Superdex 200-HR. The column was calibrated with the following standard proteins: -amylase (200 kDa), alcohol dehydrogenase (150 kDa), aldehyde dehydrogenase 3 (100 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa).

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BENZOIC ACID CARBOXYL METHYLTRANSFERASE149TABLE IIStability of the enzyme. The puried proteins (both from petal tissue and from E. coli) were highly stable for several months when stored at 80°C. When stored in Buffer A at 4°C, BAMT protein was stable for 1 week. BAMT was 100% stable for 30 min at 20°C and 60% stable for 30 min at 30°C. It was 20% stable for 30 min at 42°C but after 30-min incubation at 65°C, it completely lost activity (not shown). Kinetic properties. Kinetic studies were performed using puried plant and E. coli expressed BAMT proteins. The BAMT cDNA contains a second methionine codon two codons downstream of the rst ATG codon (at position 4 in the protein). Since the N-terminal sequencing of the protein was unsuccessful due to a blocked N-terminus, we were not able to determine the methionine used as the translational initiation site in planta. However, comparisons of the nucleotide sequence around these two ATG codons with the consensus sequences (15) as well as the fact that in the majority of characterized plant genes the rst in-frame methionine codon in the mature mRNA acts as a translational initiation site suggested that the rst ATG in the BAMT cDNA is the initiating codon. The open reading frame was therefore amplied starting with the rst methionine codon (Met 1) and ligated into the NdeI–BamHI sites of the E. coli expression vector pET28, creating an in-frame fusion with an N-terminal polyhistidine (6 His) tag. To check if BAMT protein retains its enzymatic activity when starting from the second methionine codon and, if so, to characterize its kinetic properties, the BAMT open reading frame was also amplied starting with the second methionine codon (Met 2) and ligated into the NdeI–BamHI sites of the pET-28 vector in the same way as described above. All of these constructs (and a pET-28 control plasmid without an insert) were used to transform E. coli BL21(DE3) cells, and expression of foreign gene was induced by IPTG as described under Materials and Methods. Lysates of cells carrying the BAMT constructs (Met 1 and Met 2) had substantial BAMT activity after IPTG induction (Table II). Moreover, lysates of cells expressing BAMT Met 2 protein had almost 2.4 times higher specic activity than BAMT Met 1. This is probably due to either a higher level of protein biosynthesis or stability of Met 2 BAMT in E. coli. When recombinant enzymes were puried from E. coli, specic activities of both enzymes were very similar, being 135 and 140 pkat/mg of protein for BAMT protein starting from Met 1 and Met 2, respectively (Table II). Substrate interaction kinetics have been done for puried E. coli BAMT (Met 1) protein, whereas saturation kinetics were used to measure K m for plant BAMT and E. coli BAMT (Met 2). The reaction catalyzed by BAMT exhibited Michaelis–Menten kinetics with re-Plant BAMT Gene Expression in E. coli BL21(DE3) Cells aSpecic activity (pkat/mg of protein) Construct pET-28 pET-28-BAMT Met 1 b pET-28-BAMT Met 2 c Crude lysates — 9 22 Puried protein — 135 140a Growing conditions and protein purication are described under Materials and Methods. Values are average of four independent experiments. b This construct contains the entire open reading frame of snapdragon BAMT cDNA starting with the rst methionine codon. The amount of BAMT protein produced per liter of bacterial culture was 1.6 mg. c This construct contains the entire open reading frame of snapdragon BAMT cDNA starting with the second methionine codon, two amino acids downstream of the rst one. The amount of BAMT protein produced per liter of bacterial culture was 2.7 mg.spect to its substrate saturation response. The kinetic analysis was consistent with the ordered bi– bi mechanism (Fig. 4A) reported previously for some O-methyltransferases from plants (16 –18). The BAMT (Met 1) puried from E. coli had apparent K m values for benzoic acid and SAM of 1.5 mM and 87 M, respectively. For plant-puried BAMT, the respective K m values were determined to be 1.1 mM and 28 M. K m values for E. coli BAMT (Met 2) were the same as for E. coli BAMT (Met 1) protein (Table III). K m values for benzoic acid were found to be unusually high when compared with K m values of plant O-methyltransferases for the phenolic substrates; however, similar K m values for substrates have also been found for other plant methyltransferases (18, 19). Product inhibition kinetics were used to determine the reaction mechanism of BAMT protein. The last product to be released would act as a competitive inhibitor to the rst substrate to bind and as a noncompetitive inhibitor to the second substrate. S-AdenosylL-homocysteine (SAH) is generally considered to be a potent inhibitor of plant methyltransferases (20). Product inhibition analysis revealed that inhibition by SAH was competitive with respect to SAM (Fig. 4B) and noncompetitive with respect to BA (Fig. 4C). The K i value of SAH was determined to be 7 M for SAM and 14 M for BA. Since the inhibition by SAH was competitive with respect to SAM and noncompetitive with respect to BA, SAM appears to be the rst substrate to bind to the enzyme. The methylated product, methyl benzoate, would be the rst to be released and SAH the last. This pattern was consistent with the ordered bi– bi mechanism whereby the product of the last substrate to bind to the enzyme is the rst to be released (21).

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150MURFITT ET AL.oral scent production in Antirrhinum revealed that production of methyl benzoate in petal tissue of snapdragon owers is regulated by the amount of benzoic acid and by the amount of BAMT protein, which, in turn, is regulated at the transcriptional level (10). The concentration of free BA in petal tissue varies from 2 mM on the second day of anthesis to 0.2 mM on the 12th day after anthesis (10). Since the level of BA in cells is in its K m value range, BAMT activity in snapdragon owers during ower development is most likely regulated by availability of BA rather than the SAM/SAH concentration ratios. BAMT from snapdragon and SAMT from C. breweri (8) dene a new class of carboxyl methyltransferases. These enzymes have in common the ability to transfer the methyl group of SAM to a free carboxyl group of salicylic and benzoic acids with formation of methyl salicylate and methyl benzoate, respectively. SAMT is highly specic for salicylic acid but it does methylate benzoic acid although its K m value for BA is much higher (8). In contrast, BAMT can use only benzoic acid as substrate. Both enzymes are active as dimers, with a subunit molecular mass of 40.3 and 41 kDa, respectively. When the properties of BAMT are compared with those of SAMT, it is found that SAMT and BAMT are very similar with respect to their K m values for SAM (9 and 28 M, respectively). However, the K m value of BAMT for BA (1.1 mM) is much greater than K m values of SAMT for SA and BA (24 and 190 M, respectively) (8). Plant O-methyltransferases known today constitute a distinct superfamily whose members share similarity at common conserved domains that are likely to be involved in SAM and metal binding (9, 24). Comparisons of the predicted amino acid sequences of BAMT and SAMT showed that they do not share any signicant similarity to previously characterized proteins, including other plant O-methyltransferases (8, 10). Isolation and characterization, including structuralFIG. 4. Kinetic analysis of E. coli BAMT protein. (A) Double-reciprocal plots of initial velocities with SAM varied in the presence of different concentrations of BA. Product inhibition: (B) inhibition of methylation reaction by SAH with respect to SAM (the concentration of BA was xed at 4 mM); (C) inhibition of methylation reaction by SAH with respect to BA (the concentration of SAM was xed at 150 M).TABLE IIIKinetic Parameters of Puried Plant and E. coli Expressed BAMT ProteinsKinetic parameters aKinetics properties of BAMT suggest the way in which the enzyme may be regulated in plants. Low afnities for BA (K m 1.1 mM) and moderate afnities for SAM (K m 28 M) with high levels of inhibition by SAH (K i 14 M) indicate that BAMT activity may be regulated by the intracellular SAM/SAH concentration ratio rather than BA availability. The involvement of SAM/SAH concentration ratios in controlling methyltransferase activities in vivo has been shown in several legumes (17, 22, 23). However, our recent research onBAMT (origin) Plant E. coli Met 1 E. coli Met 2K m(BA) (mM) 1.1 1.5 1.6K m(SAM) ( M) 28 87 78V max (pkat/mg) 220 300 300k cat (s 1) b 0.02 0.03 0.03a Replots of data from substrate interaction, saturation, and product inhibition experiments were used to determine the value of the kinetic parameters. b k cat, turnover number of enzyme.

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