MBE Advance Access originally published online on March 20, 2007
Molecular Biology and Evolution 2007 24(6):1320-1329; doi:10.1093/molbev/msm053
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Research Articles |
Positive Selection for Single Amino Acid Change Promotes Substrate Discrimination of a Plant Volatile-Producing Enzyme
Department of Biological Sciences, Western Michigan University
E-mail: todd.barkman{at}wmich.edu.
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Abstract |
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We used a combined evolutionary and experimental approach to better understand enzyme functional divergence within the SABATH gene family of methyltransferases (MTs). These enzymes catalyze the formation of a variety of secondary metabolites in plants, many of which are volatiles that contribute to floral scent and plant defense such as methyl salicylate and methyl jasmonate. A phylogenetic analysis of functionally characterized members of this family showed that salicylic acid methyltransferase (SAMT) forms a monophyletic lineage of sequences found in several flowering plants. Most members of this lineage preferentially methylate salicylic acid (SA) as compared with the structurally similar substrate benzoic acid (BA). To investigate if positive selection promoted functional divergence of this lineage of enzymes, we performed a branch-sites test. This test showed statistically significant support (P < 0.05) for positive selection in this lineage of MTs (dN/dS = 10.8). A high posterior probability (pp = 0.99) identified an active site methionine as the only site under positive selection in this lineage. To investigate the potential catalytic effect of this positively selected codon, site-directed mutagenesis was used to replace Met with the alternative amino acid (His) in a Datura wrightii floral–expressed SAMT sequence. Heterologous expression of wild-type and mutant D. wrightii SAMT in Escherichia coli showed that both enzymes could convert SA to methyl salicylate and BA to methyl benzoate. However, competitive feeding with equimolar amounts of SA and BA showed that the presence of Met in the active site of wild-type SAMT resulted in a >10-fold higher amount of methyl salicylate produced relative to methyl benzoate. The Met156His-mutant exhibited little differential preference for the 2 substrates because nearly equal amounts of methyl salicylate and methyl benzoate were produced. Evolution of the ability to discriminate between the 2 substrates by SAMT may be advantageous for efficient production of methyl salicylate, which is important for pollinator attraction as well as pathogen and herbivore defense. Because BA is a likely precursor for the biosynthesis of SA, SAMT might increase methyl salicylate levels directly by preferential methylation and indirectly by leaving more BA to be converted into SA.
Key Words: positive selection • enzyme evolution • SAMT • methyl salicylate
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Plant secondary metabolism is responsible for the production of molecules important for many types of ecological interactions and has likely played a critical role in the historical diversification and rise to dominance of angiosperms on land. In spite of the importance of these compounds, only rarely has the phylogenetic context promoting secondary chemical–producing enzyme functional divergence been experimentally investigated (Barkman 2003
; Benderoth et al. 2006). In the past 10 years, remarkable progress has been made toward the isolation and characterization of the enzymes responsible for volatile secondary compound biosynthesis in plants (Pichersky et al. 2006). In both reproductive and vegetative tissues of angiosperms and gymnosperms, the genes responsible for the production of terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives have been isolated (Dudareva et al. 2000; Pichersky and Gershenzon 2002; Dudareva et al. 2004; Pichersky et al. 2006). Although most of the genes for mono- and sesquiterpenoid biosynthesis appear to belong to a single gene family with several subfamilies (Bohlmann et al. 1998, 2000; Lu et al. 2002; Dudareva et al. 2003), the genes involved in volatile phenylpropanoid biosynthesis belong to members of disparate gene families (Wang and Pichersky 1998; Nam et al. 1999; Beekwilder et al. 2004; Effmert et al. 2005). Several surprising results have emerged from these studies. First, although most volatile-producing enzymes appear specialized to predominantly form a single product, many enzymes can catalyze reactions involving different substrates, resulting in the potential of a single enzyme to produce multiple products. For instance, farnesoic acid carboxyl MTs can catalyze methyl transfer to farnesoic, geranic, and lauric acid (Yang et al. 2006). Second, some enzymes (particularly those involved in terpenoid biosynthesis) can catalyze the production of multiple different volatile products from a single precursor molecule. Specifically, both longifolene and (–)-
/ß-pinene synthase can produce more than 7 products from a single substrate, although usually there are a few major products with several minor products (Martin et al. 2004). Third, distantly related enzymes have convergently evolved to produce identical volatile compounds. Eugenol O-methyltransferase (EOMT) and isoeugenol O-methyltransferase (IEMT) are highly divergent in primary sequence, yet both catalyze the formation of methyl eugenol (Wang and Pichersky 1998; Gang et al. 2002). Thus, plant volatile-producing enzymes provide an excellent opportunity to examine the evolution of protein functional divergence.
The SABATH gene family of methyltransferases (MTs) is composed of functionally diverse members that have evolved substantial biochemical specialization. Although not all members of the family have been functionally characterized, 8 MTs have been identified: jasmonic acid methyltransferase (JMT) (Seo et al. 2001), farnesoic acid methyltransferase (FAMT) (Yang et al. 2006), indole acetic acid methyltransferase (IAMT) (Zubieta et al. 2003), benzoic acid methyltransferase (BAMT) (Murfitt et al. 2000), salicylic acid methyltransferase (SAMT) (Ross et al. 1999), benzoic acid/salicylic acid methyltransferase (BSMT) (Pott et al. 2004), theobromine synthase (TCS), and caffeine synthase (CTS) (D'Auria et al. 2003). Within this gene family, the ability to catalyze the methylation of multiple substrates has been shown for several enzymes, including BSMT and FAMT; however, most of the enzymes in this family appear to be specialized and predominantly methylate a single substrate for which they are named. Although some of the divergent gene family members have evolved specialization with structurally different substrates, such as jasmonic acid and indole acetic acid, others have specialized to methylate structurally similar substrates like salicylic acid (SA) and benzoic acid (BA) that differ only by a hydroxyl group (fig. 1). The evolutionary and molecular basis for the biochemical specialization is not clear, but it is interesting that BAMT from Antirrhinum exhibits no activity with SA (Murfitt et al. 2000) and conversely SAMT from most species exhibits relatively low activity with BA (Effmert et al. 2005). In this paper, we investigate the evolution of substrate specialization of SAMT.
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SAMT sequences have been isolated from diverse plant lineages. In Clarkia breweri (Onagraceae) (Ross et al. 1999
), Stephanotis floribunda (Apocynaceae) (Pott et al. 2002), and various Solanaceae (Negre et al. 2003; Martins et al. forthcoming), SAMT is expressed at high levels in petal tissues and is involved in the formation of methyl salicylate (MeSA) and/or methyl benzoate (MeBA) as floral scent compounds (fig. 1). In the case of Antirrhinum majus (Scrophulariaceae) (Negre et al. 2002) and Atropa belladonna, as well as numerous other genera of Solanaceae (Fukami et al. 2002; Ament et al. 2004; Martins and Barkman 2005), SAMT may be involved in defense or cellular detoxification. All SAMT sequences that have been biochemically characterized have the lowest Km and highest Kcat/Km with SA, although they have activity with other substrates like BA (Effmert et al. 2005). Recently, sequences shown to be similar to SAMT have been isolated from Nicotiana suaveolens (Pott et al. 2004), Petunia hybrida (Negre et al. 2003), and other Solanaceae (Martins et al. forthcoming) floral tissues. These genes, named BSMT, encode an enzyme that catalyzes the formation of both MeSA and MeBA. BSMT from Petunia is much more efficient at converting SA to MeSA, whereas BSMT from N. suaveolens most efficiently converts BA to MeBA in vitro. However, the activities of both enzymes result in the emission of MeBA from flowers. A crystal structure of C. breweri SAMT was recently solved and used to identify the active site of the enzyme, including the substrate-binding amino acids (Zubieta et al. 2003). Site-directed mutagenesis of several of these active site residues resulted in alteration of the substrate specificity of SAMT, such that it showed higher activity with jasmonic acid as compared with SA. Although these sites have been shown to affect the substrate specificity of SAMT, the evolutionary context for functional divergence of the enzyme remains unclear. Because SAMT is well characterized and exhibits a relatively high degree of substrate specialization, we investigated whether there has been adaptive evolution of this enzyme for the efficient production of MeSA.
A hypothesis of positive selection for enzyme evolution relies on adaptive roles of the products formed as a result of enzyme activity. In the case of SAMT, MeSA has many functions, all with probable impacts for fitness. MeSA may have an important role for pollination of C. breweri and Datura wrightii because both emit the volatile and express SAMT in floral tissues (Ross et al. 1999; Martins et al. forthcoming). Furthermore, electroantennogram studies indicate a large physiological response of their hawk moth pollinators to this compound (Raguso et al. 1996; Raguso and Light 1998; Fraser et al. 2003). In addition to plant–pollinator interactions, MeSA has been shown to be a mediator of plant–pathogen interactions. Studies of tobacco have shown that MeSA, synthesized from endogenous SA, acts as an exogenous volatile signal during pathogen infection, which appears to stimulate systemic acquired resistance in uninfected parts of the same plant as well as uninfected neighboring plants (Shulaev et al. 1997; Seskar et al. 1998; Deng et al. 2004). In accordance with a pathogen defense role, plant tissues treated with SA, the hallmark chemical produced in response to pathogen inoculation, show increased SAMT expression relative to untreated tissues (Fukami et al. 2002; Negre et al. 2002). In response to herbivory, MeSA has been detected as part of a blend of volatiles emitted from herbivore damaged leaves (Dicke et al. 1990; Poecke et al. 2001). This volatile may serve as an attractant to parasitoids of caterpillars feeding on the leaves, thus alleviating the plant of herbivore-induced stress. Consistent with a function for herbivore defense, increased SAMT expression has been shown to be associated with MeSA emission in spider mite–damaged leaves in tomato (Ament et al. 2004). The fact that MeSA may have an important role in plant reproduction and survival suggests that the efficient production of MeSA by SAMT is important for plant fitness. MeBA, the other product formed by SAMT (as well as BSMT), is also likely involved in plant–pollinator interactions, and it elicits a physiological and behavioral responses in hawk moths (Raguso et al. 1996; Raguso and Light 1998; Hoballah et al. 2005); however, it has not been implicated in pathogen or herbivore defense. Below, we test the hypothesis that functional divergence of SAMT was driven by positive selection for increased efficiency of MeSA production using a combined computational and experimental approach. First, a phylogenetic analysis of the SABATH gene family was performed to identify potential sites under selection. Second, site-directed mutagenesis was used to test the functional importance of the positively selected sites.
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Materials and Methods |
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Data Analyses
DNA sequences from all enzymatically characterized SABATH gene family members were obtained from GenBank or were generated as part of this study (GenBank accession numbers EF472972 [GenBank] –EF472978 [GenBank] ). DNA sequences were aligned with ClustalX (Thompson et al. 1997
) with subsequent minor adjustments to preserve codon structure. Maximum likelihood (ML) analyses, assuming the Hasegawa–Kishino–Yano (HKY) + invariable sites + gamma (HKY + I + G) model of nucleotide substitution as chosen by Modeltest (Posada and Crandall 1998), were performed with PAUP* (Swofford 2003) using 10 random addition sequences and tree bisection-reconnection (TBR) swapping. Bootstrapping was performed using 100 replicates. PAML version 3.15 (Yang 2000) was used to test the hypothesis of positive selection in the SABATH gene family. The branch-sites test was implemented because it is expected that positive selection should act only on a subset of sites and branches of a gene tree as functional divergence occurs. In addition, it has previously been shown that another gene required for volatile production (IEMT) has experienced positive selection using the branch-sites model (Barkman 2003).
Gene Isolation and Cloning
In order to obtain SAMT cDNA sequence from D. wrightii, RNA was isolated from petal tissue of this species because it is known to produce MeSA and MeBA as floral scent compounds (Raguso et al. 2003; Martins et al. forthcoming). Tissue samples were flash frozen in liquid nitrogen and subsequently ground to a fine powder. Total RNA was extracted using the RNeasy Plant Mini Extraction kit (Qiagen Inc, Valencia, CA) following the manufacturer's protocol. After extraction, RNA was eluted in 40 µl of RNase-free water and stored at -80 °C.
The 5' and 3' RACE were used to characterize the complete coding sequence of D. wrightii SAMT. Internal primers (Martins and Barkman 2005) were used in combination with the SMART cDNA synthesis kit (Stratagene, Cedar Creek, TX). Primers were subsequently designed to amplify the entire coding sequence. The Superscript One-Step RT-PCR Kit with Platinum Taq (Invitrogen, Carlsbad, CA) was used for all reverse transcriptase–polymerase chain reaction (RT-PCRs). Each reaction consisted of 25 µl of 2 x Reaction Mix, 1 µl of each primer (10µM), 1 µl of RT-Taq Mix, 0.5 µg of RNA, and enough sterile RNase-free water to reach a 50 µl final volume. The cDNAs were amplified under the following conditions: one cycle of cDNA synthesis at 50 °C for 30 min followed by cDNA denaturation at 94 °C for 2 min. Cycling then proceeded to denaturation at 94 °C for 45 s, annealing at 50 °C for 1 min, and extension for 1 min at 72 °C, for 40 cycles. Thermalcycling concluded with a 30 min extension at 72 °C. Following amplification, the cDNA product was ligated into a pCR/CT-TOPO vector using the manufacturer's protocol (Invitrogen Corp.). This vector was subsequently transformed into top 10 cells. Fifty microliters of the cloning reaction was plated on Luria-Bertani (LB)-agar containing 100 µg/ml of ampicillin and grown for 16 h at 37 °C. In order to determine the correct orientation of SAMT-containing clones, we used a polymerase chain reaction (PCR) screening method. Once sense colonies were identified and determined to be Taq error free, minipreps from a 3 ml overnight culture were performed using the Qiagen plasmid mini kit following the manufacturer's protocol.
Site-Directed Mutagenesis
The Stratagene QuickChange kit was used to change the codon under positive selection in D. wrightii with the following 2 mutagenic primers that matched floral–expressed SAMT except for the single codon that changed ATG to CAC. Coding strand: 5'-GTTCACTCCTCTTATAGTCTCcacTGGCTATCTCAAGTTCCTG-3' Noncoding strand: 5'-CAGGAACTTGAGATAGCCAgtgGAGACTATAAGAGGAGTGAAC-3'. Miniprepped plasmid was used as PCR template following the manufacturer's protocol. Plasmid transformation was performed following DpnI digestion of the template. Colonies were screened by PCR for full-length fragments, which were subsequently sequenced and checked for the presence of the desired mutation. Minipreps were performed to isolate the mutant plasmid for protein expression as described above.
Heterologous Expression and Gas chromatography-mass spectrometry Analysis
Protein expression was driven by the T7 promoter following the protocol found in the pCRT7 TOPO TA Expression Kit. Because the insert encoded a stop codon, the His tag was not expressed. Plasmids containing the insert of interest were transformed into BL21 (DE3)pLysS cells. The culture was grown to optical density (OD) 0.5–0.8 prior to addition of isopropyl-L-thio-ß-galactoside (IPTG). Once IPTG was added, cells were allowed to grow at 37 °C with shaking for 1.5 h before addition of substrates. We provided 1.4 µmol of SA only, BA only, or a combination of 1.4 µmol of SA and BA. These sets of substrate concentrations were chosen in order to directly assess relative preference of the mutant and wild-type enzyme for these 2 substrates and represent the range of SA and BA substrate ratios reported for plants (Ribnicky et al. 1998; Effmert et al. 2005). In order to assess enzyme preference for either SA or BA, enzyme assays needed to be performed with saturating concentrations of substrate. Our initial studies demonstrated that 1.4 µmoles of substrate were saturating because even the addition of 7 µmoles of substrate did not result in higher levels of product formation over the time period studied here. Following a 15-min incubation with the substrates, cells were pelleted and the supernatant (spent growth medium) collected. Volatiles were extracted from spent growth medium with 5 ml Hexane and the recovered hexane phase was transferred to a vial and concentrated to 200 µl. Negative controls included 50 ml growths in which an antisense vector was used for transformations as well as those that had either substrate or plasmid omitted. This basic procedure is a modification of Ross et al. (1999). Replicate transformations were performed and the amount of product from each was determined. Gas chromatography-mass spectrometry (GC-MS) analyses were performed on an HP6890 GC System coupled to an HP5973 Mass Selective Detector using a DB-5 capillary column. The oven conditions were 40 °C for 2 min, ramping 20 °C/min to 300 °C with a 2 min hold. Toluene was used as an internal standard for quantitation.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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ML phylogenetic analyses of 14 functionally characterized SABATH gene family members resulted in a single tree (–ln L = 14510.77) (fig. 2). Functionally characterized SABATH family members have been isolated from only a single species in most cases. However, SAMT has been isolated from several flowering plant families and appears to be monophyletic with relatively high bootstrap support (BP = 92). Two BSMT sequences from N. suaveolens and P. hybrida appear to be nested within the SAMT lineage; however, this close relationship is not surprising given that BSMT can catalyze the formation of MeSA as well as MeBA (Negre et al. 2003
; Pott et al. 2004). Thus, enzymes that catalyze the formation of MeSA appear to be monophyletic, with the exception of BSMT from Arabidopsis thaliana/Arabidopsis lyrata, which are as distantly related to the SAMT lineage as any other gene family member. No SABATH gene family member within the Arabidopsis genome is included in the SAMT lineage (D'Auria et al. 2003). Therefore, if A. thaliana historically possessed a sequence orthologous to SAMT, it must have been lost. Thus, as previously reported, BSMT from Arabidopsis likely evolved the ability to synthesize MeSA and MeBA by convergent evolution relative to SAMT (Chen et al. 2003).
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Given the monophyly of SAMT genes, a likelihood ratio test (LRT) was performed to test whether positive selection may have promoted the enzymatic divergence of SAMT from the other related MTs. Because we are especially interested in the evolution of the ability to synthesize MeSA, our analyses focused on the branch separating SAMT from all other SABATH gene family members. The branch-sites model developed in Yang and Nielsen (2002) relies on the a priori specification of foreground and background lineages. In this case, the foreground lineage is the branch separating the SAMT lineage from all others (shown in bold, fig. 2), whereas the background lineages are all other branches in the tree. Proportions of sites belonging to each of the 2 site classes (
0 and 1) are estimated for all lineages and a 3rd site class (2) is estimated for the foreground lineage alone. The null hypothesis has the same parameters except that 2 is set to 1 (Zhang et al. 2005). A significant LRT for this model comparison would indicate that sites in the foreground lineage are under positive selection if 2 is greater than 1 (Yang and Nielsen 2002). The LRT statistic from the analysis of the SABATH sequence data was 2(diff. ln L) = 4.1 (P = 0.04, 1 degree of freedom). In the branch-sites model, 66.4% of sites were estimated with 0 = 0.14, 31.9% with 1 = 1.0, and 1.7% with 2 = 10.8. These results suggest that there has been strong positive selection on a small number of sites along the branch separating the SAMT lineage from all other SABATH gene family members.
Assuming the branch-sites model, PAML 3.15 (Yang 2000) was used to calculate the posterior probability that a particular site belongs to the site class that has experienced positive selection using the Bayes empirical Bayes method (Yang et al. 2005). Only a single site was inferred to have experienced positive selection with high probability: amino acid 156 changed from His to Met along this branch and the posterior probability is 0.99 that it is in the site class characterized by 2. This site has been identified as an active site residue from the SAMT crystal structure of C. breweri (Zubieta et al. 2003). One other site, amino acid 336, had a low posterior probability of 0.56 and is not predicted to be within the active site of SAMT so we did not evaluate it further. Because the test indicates that site 156 was positively selected for replacement, we performed an experiment to test for effects of this site on enzyme catalysis. Specifically, we hypothesized that the active site Met would confer an ability of SAMT to discriminate among different substrate types. In this study, we evaluated the preference of SAMT for the methylation of SA relative to BA. The biosynthesis of SA likely occurs by the direct conversion of BA by the activity of benzoic acid 2-hydroxylase (B2H) (fig. 1). Therefore, cells producing SA should produce BA first (Ribnicky et al. 1998). In this case, it is expected that the ability of SAMT to discriminate between the 2 available substrates would be advantageous particularly if efficient production of MeSA, but not MeBA, is important for organismal fitness.
A 1086-bp full-length SAMT sequence was isolated from D. wrightii floral tissue. This sequence was subsequently cloned into the TOPO TA expression vector. Initial experiments indicated that this sequence could catalyze the formation of MeSA when SA was supplied to the growth medium (Martins et al. forthcoming). In order to test for the function of the putative positively selected site in SAMT, we mutated the selected codon that codes for Met in D. wrightii (and all other characterized SAMT sequences) to His. We provided the heterologously expressed enzymes with equimolar concentrations of SA and BA in order to determine whether SAMT is capable of distinguishing between these 2 structurally similar substrates. As shown in figure 3A, wild-type SAMT from D. wrightii produces very low amounts of MeBA relative to MeSA even though equimolar amounts of the substrates BA and SA were supplied to the growth medium. In contrast, Met156His SAMT produced nearly equal amounts of MeSA and MeBA when provided with equimolar amounts of substrates (fig. 3B).
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Figure 4 shows quantitative results of the wild-type and mutant enzyme assays. As shown in figure 4A, D. wrightii wild-type SAMT can produce MeBA when provided only BA in the growth medium. However, when the medium was supplemented with equimolar amounts of SA and BA, very little MeBA was produced. In fact, the amount of MeBA produced relative to MeSA when both substrates were present was indistinguishable from the relative amounts of product formed when only SA was added. In contrast, figure 4B shows that D. wrightii mutant SAMT produced nearly equal molar amounts of MeBA and MeSA when both substrates were provided. Single substrate feeding experiments showed that the mutant sequence can produce both MeBA and MeSA at similar levels when only single substrates were provided. Both the wild-type and mutant enzymes catalyzed the formation of low levels of MeBA, even though no BA or SA was added to the growth medium. This is likely due to the release of BA by metabolism of the LB medium. However, it is not due to an inherent ability of Escherichia coli to catalyze the formation of MeBA because neither nontransformed BL21 cells nor SAMT antisense-transformed cells could catalyze the formation of either MeBA or MeSA (results not shown). Replication of the double substrate feeding experiment (n = 4) showed that wild-type D. wrightii exhibits a 14-fold higher level of MeSA production as compared with MeBA, whereas the mutant showed a less than 2-fold higher level of MeSA production as compared with MeBA (fig. 4C).
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Because both SAMT and BSMT have been isolated from Solanaceae (Fukami et al. 2002
; Negre et al. 2003; Martins and Barkman 2005; Martins et al. forthcoming), we investigated active site evolution between these enzymes within this plant family. As shown in figure 5, phylogenetic analysis of SAMT and BSMT (excluding the active site codon 156) from several members of Solanaceae resulted in a single tree (–ln L = 12931.72). It appears that an ancestral duplication has resulted in 2 well-supported, closely related lineages of sequences in the family (Martins et al. forthcoming). One lineage comprised Met-containing SAMT sequences that are more efficient at methylating SA than BA (Negre et al. 2003), whereas the other comprised His-containing BSMT sequences that appear to be more efficient at methylating BA than SA (Pott et al. 2004).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Plant secondary metabolism has such a central role in a multitude of ecological interactions that the evolution of efficient enzyme catalysis should be predicted to improve organismal fitness. This study documents a historical episode of positive selection on SAMT, which may have promoted its functional divergence. The branch-sites model detected strong positive selection (
2 = 10.8) on a single codon within the active site of SAMT along the branch separating the ancestral sequence from all other SABATH family members. This site is a highly conserved His in 20 out of 24 other family members in the genome of Arabidopsis as well as all other functionally characterized members of the SABATH gene family, but only in SAMT sequences is it replaced by Met. Based on the results of our site-directed mutagenesis study, it appears that this strong selection may have promoted the acquisition of a >10-fold ability of this enzyme to discriminate between the structurally similar, biosynthetically related substrates, SA and BA. Future studies should investigate the degree to which the active site Met promotes discrimination among other potential substrates as well. The crystal structure of SAMT suggests that this Met is part of the active site and it, along with other active site residues, produces a narrow substrate-binding pocket that likely accommodates the planar conformation of SA (Zubieta et al. 2003). The fact that BA has axis of rotation by the carboxyl group makes this molecule a dynamic substrate that does not appear to be accommodated effectively in the narrow Met-containing pocket (Zubieta et al. 2003).
The effect of the replacement of Met with His in D. wrightii SAMT is consistent with kinetic parameter differences estimated for all characterized SAMT relative to BSMT (Ross et al. 1999; Negre et al. 2002; Chen et al. 2003; Pott et al. 2004; Effmert et al. 2005). A comparison of Met-containing SAMT and His-containing BSMT enzymes, suggests that the apparent preference for SA by SAMT is not reflected in Km for that substrate because the range of values are similar for the 2 enzymes (24–250 µM and 16–162 µM, respectively). Instead, SAMT and BSMT differ substantially in their affinities for BA. The range of Km for this substrate is 190–2900 µM for SAMT and 65–149 µM for BSMT. This difference in affinity for BA between these enzymes is also apparent upon consideration of their average Km SA/Km BA (SAMT: 19.84 ± 10.3 vs. BSMT: 1.97 ± 1.8). This biochemical divergence is consistent with the results of our experimental studies and implicate Met as the primary determinant of the substrate specialization of SAMT.
Direct comparisons of our experimental results to previous site-directed mutagenesis studies of C. breweri SAMT that converted Met to His are difficult to make mostly because Zubieta et al. (2003) generated mutants with multiple amino acid changes. However, it should be noted that, like our Met156His SAMT, all C. breweri SAMT mutants that had the active site Met changed to His showed less preference for SA as compared with other substrates relative to wild-type enzyme. Evolution of substrate discrimination among other SABATH family members likely involves other amino acids than the Met mutated here so that there is probably not a single active site residue governing substrate specificity of all the divergent SABATH enzymes. As shown for TCS, mutagenesis of site 221 from His to Arg partly changed the substrate specificity of this enzyme so that it had increased CTS activity (Yoneyama et al. 2006). Molecular evolutionary analyses should be performed for the rest of the gene family members in order to elucidate the selective context for functional divergence in general.
The evolution of enzymatic discrimination of structurally related compounds is likely to be a general challenge to plant enzymes involved in secondary metabolism due to the fact that potential substrates are often produced in the same biochemical pathway via sequential modifications of structurally similar precursors. Therefore, adaptive evolution for the efficient production of important chemicals may be predicted for many enzymes involved in secondary metabolism. Indeed, like SAMT, previous studies of IEMT revealed positive selection driving amino acid replacements that promoted a change in substrate specificity of that volatile-producing enzyme (Barkman 2003). IEMT catalyzes the methylation of eugenol and isoeugenol while showing little activity towards the structurally related substrates caffeic and hydroxyferulic acid (Wang and Pichersky 1998; Wang and Pichersky 1999). In the case of SAMT, the ability to discriminate between SA and BA may be necessary in planta in order to predominantly produce MeSA. SA appears to be produced from BA by the activity of B2H (Lee et al. 1995; León et al. 1995; Ribnicky et al. 1998; Boatright et al. 2004), although an alternative pathway from isochorismate has been described (Wildermuth et al. 2001) (fig. 1). This biochemical dependence of SA production on the prior synthesis of BA could present a biochemical conundrum for plants for which the efficient production of MeSA, but not MeBA, is important for pollination or defense. Yet, although SAMT may be exposed to both substrates simultaneously in plant cells, active site evolution seems to have led to effective substrate discrimination (fig. 3A and 4). It is not entirely clear whether the evolution of Met in the active site results in an increased ability to produce MeSA or a decreased ability to synthesize MeBA or some combination of both. Future studies need to investigate purified mutant and wild-type enzymes to discern whether replacement of His by Met results in changes in Km and Kcat of the enzyme for both substrates. The challenge of discriminating between structurally similar substrates involved in primary metabolism has also been recognized for rubisco and its competing substrates CO2 and O2 (Yu et al. 2005) as well as for aminoacyl-tRNA synthetases that need to distinguish between similar amino acids (Prætorius-Ibba et al. 2000).
The presence of both paralogous SAMT and BSMT genes within Solanaceae that can perform similar biochemical transformations may allow for subfunctionalization or perhaps even neofunctionalization (fig. 5). In N. suaveolens, BSMT is expressed highly within flowers but not other tissue types (Pott et al. 2004) where it appears responsible for MeBA emission in floral scent. SAMT, on the other hand, is upregulated in leaves in response to SA treatment (Fukami et al. 2002; Martins and Barkman 2005). Yet, in Petunia, the Met-containing BSMT (which is part of the SAMT lineage of fig. 5) is expressed in flowers where it predominantly produces MeBA (Negre et al. 2003) and in D. wrightii, both SAMT and BSMT are expressed in floral tissues where both MeSA and MeBA are produced (Martins et al. forthcoming). The evolutionary lability of this active site residue in SAMT and BSMT is surprising because the codon for Met (AUG) is highly divergent from His (CAC and CAU). Yet, if our data and previous active site modeling are indicative (Zubieta et al. 2003; Effmert et al. 2005), the evolutionary reversal to His of Solanaceae BSMT may permit some degree of active site flexibility. In fact, heterologous expression of a BSMT sequence from N. suaveolens indicates that it can produce methyl nicotinate in addition to MeSA and MeBA (data not shown). Although Met and His codons are divergent at all the 3 codon positions, 1 codon for Glu is intermediate between the 2 (CAG) and could provide an advantageous intermediate active site residue. In fact, Schizanthus (Solanaceae) SAMT does encode Glu using CAA at position 156 (Martins and Barkman 2005) as do several sequences available on GenBank that are closely related to SAMT (data not shown). We are currently investigating the effect of a Glu replacement on SAMT substrate preference.
The use of site-directed mutagenesis to complement molecular evolutionary analyses is a powerful approach that can provide insight into the evolution of enzyme functional divergence, particularly when guided by evolutionarily motivated hypotheses. Our confidence in the role of the active site Met of SAMT in discriminating between these 2 structurally similar substrates, SA and BA, is high, particularly because of the agreement among evolutionary, crystal structure, and mutagenesis studies. Although termed "secondary" chemicals, it would appear that many of these compounds play important roles in the life history of the organisms producing them. Because both SAMT and IEMT are involved in floral scent production they may have direct roles in sexual reproduction in plants. Therefore, as is the case for the majority of positively selected genes in animals, studies of SAMT and IEMT reinforce the notion that genes enhancing the reproductive fitness of an organism may experience adaptive evolution, even for one or a few amino acids that have major impacts on enzyme function.
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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We thank Doug Coulter, Don Kane, Maarten Vonhof, Cathy Foune, and Pam Hoppe for helpful discussions and/or assistance. Eran Pichersky provided valuable technical suggestions. Two anonymous reviewers provided helpful suggestions to improve the manuscript. The Chemistry Department at Western Michigan University is also thanked for allowing us to use the GC-MS for volatile analyses. This study was supported by National Science Foundation grant DEB 0344496, a National Institutes of Health-Bridges research assistantship, and the Western Michigan University Post-Baccalaureate Scholars Program.
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Footnotes |
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1 Present address: Department of Botany, University of Wisconsin
Jianzhi Zhang, Associate Editor
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References |
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