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Eran Pichersky - One of the best experts on this subject based on the ideXlab platform.
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2002. Purification and characterization of benzoate:coenzyme A ligase from Clarkia breweri
2015Co-Authors: Till Beuerle, Eran PicherskyAbstract:Benzoate:CoA ligase (BZL) was partially purified from flowers of the annual California plant Clarkia breweri. BZL catalyzes the formation of benzoyl-CoA and anthraniloyl-CoA, important intermediates for subsequent acyltransferase reactions in plant sec-ondary metabolism. The native enzyme is active as a monomer with a molecular mass of 59–64.5 kDa, and it has Km values of 45, 95, and 130 lM for benzoic acid, ATP, and CoA, respectively. BZL is most active in the pH range of 7.2–8.4, and its activity is strictly dependent on certain bivalent cations. BZL is an AMP-forming enzyme. Overall, its properties suggest that it is related to the family of CoA ligase enzymes that includes the plant enzyme 4-hydroxycinnamate:CoA ligase. 2002 Elsevier Science (USA). All rights reserved
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Biosynthesis of t-Anethole in Anise: Characterization of t-Anol/Isoeugenol Synthase and an O-Methyltransferase Specific for a C7-C8 Propenyl Side Chain 1(W)(OA)
2009Co-Authors: Takao Koeduka, Joseph P. Noel, Eran Pichersky, Thomas J. Baiga, Jack H. SkirballAbstract:The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl- 14 C]adenosyl-Lmethionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 mM for coumaryl acetate and 230 mM for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 mM for isoeugenol and 54.5 mM for S-[methyl- 14 C]adenosyl-L-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol. The phenylpropenes are a class of volatile compounds found throughout the gymnosperms and angiosperms. When emitted from flowers, they serve as attractants for pollinators, which detect them through their olfactory systems. In addition, at high concentrations their general toxicity to cells renders them useful as defense compounds, and consequently they are found in vegetative tissues of many plant species, although perhaps due to their toxicity they are typically sequestered in specialized structures or quickly emitted from the plant. For example, eugenol and methylchavicol are synthesized and stored in glandular trichomes on the surface of leaves of sweet basil (Ocimum basilicum, Lamiaceae; Gang et al., 2001; Iijima et al., 2004), and eugenol, isoeugenol, and their meth
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Biosynthesis of t-Anethole in Anise: Characterization of t-Anol/Isoeugenol Synthase and an O-Methyltransferase Specific for a C7-C8 Propenyl Side Chain
Plant Physiology, 2008Co-Authors: Takao Koeduka, Joseph P. Noel, Thomas J. Baiga, Eran PicherskyAbstract:The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.
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Structural, Biochemical, and Phylogenetic Analyses Suggest That Indole-3-Acetic Acid Methyltransferase Is an Evolutionarily Ancient Member of the SABATH Family
Plant Physiology, 2007Co-Authors: Nan Zhao, Joseph P. Noel, Eran Pichersky, Jeannine R. Ross, Yue Yang, Jean-luc Ferrer, Ju Guan, Feng ChenAbstract: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.
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An Arabidopsis thaliana methyltransferase capable of methylating farnesoic acid.
Archives of Biochemistry and Biophysics, 2006Co-Authors: Yue Yang, Joseph P. Noel, Eran Pichersky, Joshua S Yuan, Jeannine Ross, Feng ChenAbstract:We previously reported the identification of a new family of plant methyltransferases (MTs), named the SABATH family, that use S-adenosyl-L-methionine (SAM) to methylate a carboxyl moiety or a nitrogen-containing functional group on a diverse array of plant compounds. The Arabidopsis genome alone contains 24 distinct SABATH genes. To identify the catalytic specificities of members of this protein family in Arabidopsis, we screened recombinantly expressed and purified enzymes with a large number of potential substrates. Here,we report that the Arabidopsis thaliana gene At3g44860 encodes a protein with high catalytic specificity towards farnesoic acid (FA). Under steady-state conditions, this farnesoic acid carboxyl methyltransferase (FAMT) exhibits KM values of 41 and 71 microM for FA and SAM, respectively. A three-dimensional model of FAMT constructed based upon similarity to the experimentally determined structure of Clarkia breweri salicylic acid methyltransferase (SAMT) suggests a reasonable model for FA recognition in the FAMT active site. In planta, the mRNA levels of At3g44860 increase in response to the exogenous addition of several compounds previously shown to induce plant defense responses at the transcriptional level. Although methyl farnesoate (MeFA) has not yet been detected in Arabidopsis, the presence of a FA-specific carboxyl methyltransferase in Arabidopsis capable of producing MeFA, an insect juvenile hormone made by some plants as a presumed defense against insect herbivory, suggests that MeFA or chemically similar compounds are likely to serve as new specialized metabolites in Arabidopsis.
Robert A Raguso - One of the best experts on this subject based on the ideXlab platform.
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New Perspectives in Pollination Biology: Floral Fragrances. A day in the life of a linalool molecule: Chemical communication in a plant‐pollinator system. Part 1: Linalool biosynthesis in flowering plants
Plant Species Biology, 1999Co-Authors: Robert A Raguso, Eran PicherskyAbstract:The monoterpene alcohol, linalool, is present in the soral fragrance of diverse plant families and is attractive to a broad spectrum of pollinators, herbivores and parasitoids. Floral emission of linalool has evolved de novo in the fragrant, moth-pollinated annual Clarkia breweri (Gray) Greene (Onagraceae) through a combination of up-regulation and ectopic expression of its biosynthetic enzyme, linalool synthase (LIS), in conjunction with allometric size increases in all soral organs. Linalool synthase activity and linalool emissions are 1000-fold lower in a sibling species, C. concinna (Fischer & Meyer) Greene, that is diurnally pollinated. Linalool synthase expression is spatially and temporally regulated during C. breweri sower development, immediately precedes free linalool emission and is absent from nonsoral tissues. Its activity is highest in the style, but most of the linalool product appears to be converted to the pyranoid and furanoid linalool oxides. The LIS structural gene is a member of the terpene synthase gene family, sharing sequence identity with two discrete classes, represented by limonene synthase (LMS) and copalyl pyrophosphate synthase (CPS). Genetic crosses between C. breweri and C. concinna indicate that strong linalool emission segregates as a dominant mendelian trait, whereas the inheritance of linalool oxide formation is more complex, suggesting epistatic biosynthetic pathway interactions. We discuss areas for future research, including comparative studies of linalool biosynthesis in different plant families, entrainment of linalool emission to nocturnal circadian rhythms and the induction of vegetative linalool as an indirect herbivore defense.
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acetyl coa benzylalcohol acetyltransferase an enzyme involved in floral scent production in Clarkia breweri
Plant Journal, 1998Co-Authors: Natalia Dudareva, Robert A Raguso, Kyoung Hee Nam, John C Dauria, Eran PicherskyAbstract:Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but a 35-residue region from position 135-163 has significant similarity (42-56% identity) to several proteins known or suspected to use an acyl-CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.
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electroantennogram responses of male sphinx perelegans hawkmoths to floral and green leaf volatiles
Entomologia Experimentalis Et Applicata, 1998Co-Authors: Robert A Raguso, Douglas M LightAbstract:Electroantennograms (EAGs) from field-collected male Sphinx pereleganshawkmoths were recorded in response to 10 individual floral scent compounds identified from Clarkia breweri (Onagraceae), 21 additional volatiles characteristic of other night-blooming flowers, and eight ‘green leaf’ volatiles. Measurable EAG responses were elicited to all compounds tested, but the most effective antennal stimulants were benzyl acetate, linalool, methyl salicylate and trans-2-hexenal. Mean, pooled EAGs to oxygenated terpenoids, aromatic esters and fatty acid derivatives were larger in magnitude than those in response to aromatic aldehydes/alcohols, monoterpenes and nitrogen-bearing compounds. The rank order of male S. perelegans’ EAGs did not differ significantly from that of previously recorded responses of male Hyles lineata to the same scent compounds, and EAG magnitudes were generally larger for S. perelegans than for H. lineata. Both hawkmoth species are shown to have broad olfactory receptivities and could potentially respond to a wide array of plant volatiles as floral attractants.
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Floral scent production in Clarkia breweri. III. Enzymatic synthesis and emission of benzenoid esters
Plant Physiology, 1998Co-Authors: Natalia Dudareva, Robert A Raguso, Jihong Wang, Jeannine R. Ross, Eran PicherskyAbstract:The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.
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Acetyl‐CoA:benzylalcohol acetyltransferase – an enzyme involved in floral scent production in Clarkia breweri
The Plant Journal, 1998Co-Authors: Natalia Dudareva, Robert A Raguso, John C. D’auria, Kyoung Hee Nam, Eran PicherskyAbstract:Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but a 35-residue region from position 135-163 has significant similarity (42-56% identity) to several proteins known or suspected to use an acyl-CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.
Efraim Lewinsohn - One of the best experts on this subject based on the ideXlab platform.
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Increased substrate availability reveals the potential of scentless lisianthus flowers in producing fragrant benzenoid-phenylpropanoids.
Physiologia Plantarum, 2020Co-Authors: Fang Fang, Michele Zaccai, Moran Oliva, Rinat Ovadia, Einat Bar, Ada Nissim-levi, Varun Kumar, Ru Wang, Agmon Neeman, Efraim LewinsohnAbstract:Lisianthus (Eustoma grandiflorum), a leading plant in the cut flower industry is scentless. Here we show that lisianthus flowers have potential to produce several fragrant benzenoid-phenylpropanoids when substrate availability is not limited. To enable hyperaccumulation of substrates for the production of volatile benzenoid-phenylpropanoids, lisianthus commercial hybrid 'Excalibur Pink' was transformed via floral dipping with a feedback-insensitive E.coli DAHP synthase (AroG*) and Clarkia breweri benzyl alcohol acetyltransferase (BEAT), under constitutive promoters. The T1 progeny of 'Excalibur Pink' plants segregated into four visual phenotypes, with pink or white colored petals and multiple or single petal layers. Interestingly, transformation with AroG* and BEAT caused no significant effect in the pigment composition among phenotypes, but did increase the levels of down-stream fragrant volatile benzenoids. All the transgenic lines exclusively accumulated methyl benzoate, a fragrant benzenoid, either in their petals or leaves. Furthermore, feeding with benzyl alcohol, resulted in the accumulation of two novel benzenoids, benzyl acetate (the product of BEAT) and benzoate, as well as a dramatic increase in the concentrations of additional benzenoid-phenylpropanoid volatiles. Presumably, the degree of benzaldehyde overproduction after benzyl alcohol feeding in both leaves and flowers revealed their reverse conversion in lisianthus plants. These findings demonstrate the concealed capability of lisianthus plants to produce a wide array of fragrant benzenoid-phenylpropanoids, given high substrate concentrations, which could in turn open opportunities for future scent engineering. This article is protected by copyright. All rights reserved.
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Post-harvest enhancement of aroma in transgenic lisianthus (Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase (BEAT) gene
Postharvest Biology and Technology, 2007Co-Authors: Dina Aranovich, Efraim Lewinsohn, Michele ZaccaiAbstract:Abstract Lisianthus ( Eustoma grandiflorum ) is an ornamental plant with beautiful but scentless flowers. In an attempt to induce a fragrance in their flowers, lisianthus plants were transformed with the Clarkia breweri gene coding for benzyl alcohol acetyltransferase ( BEAT ), catalyzing the synthesis of the volatile compound benzyl acetate under the regulation of the CaMV35S promoter. An external supply of benzyl alcohol induced five to seven times higher production of benzyl acetate in detached flowers and leaves of transgenic lisianthus plants, compared to non-transformed plants. No benzyl acetate was detected in tissues of both control and transgenic plants fed with water. When fed with additional alcoholic compounds, i.e. hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol, assumed to be used as substrates by BEAT, transgenic in vitro -grown lisianthus plantlets produced significantly higher levels of acetates than control plants. These results demonstrate the possibility of producing substrate-dependent acetates in transgenic lisianthus plants, which could lead to induction of new aromas.
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Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene
Molecular Breeding, 2002Co-Authors: Michal Lavy, Efraim Lewinsohn, Amir Zuker, Olga Larkov, Uzi Ravid, Alexander Vainstein, David WeissAbstract:Most modern cut-flower cultivars, including those of carnation( Dianthus caryophyllus ), lack distinct fragrance.Carnationcv. Eilat flowers produce and emit various fragrance compounds, includingbenzoic acid derivatives and sesquiterpenes, but not monoterpenes. Based onGC-MS analysis, benzoic acid, benzyl benzoate, phenylethyl benzoate, methylbenzoate, cis -3-hexenyl benzoate and β-caryophylleneare the major fragrance compounds, representing ca. 60% of the total volatilesgenerated by these flowers. The level of these compounds increases dramaticallyduring petal development. To evaluate the possibility of producing monoterpenesin carnation cv. Eilat, we generated transgenic plants expressing the linaloolsynthase gene from Clarkia breweri under the regulation ofthe CaMV 35S constitutive promoter. The product of this gene catalyzes theproduction of the monoterpene linalool from geranyl diphosphate. HeadspaceGC-MSanalysis revealed that leaves and flowers of transgenic, but not controlplants,emit linalool and its derivatives, cis - and trans -linalool oxide. GC-MS analysis of petal extractrevealed the accumulation of trans -linalool oxide but notlinalool. The emission of linalool by the transgenic flowers did not lead todetectable changes in flower scent for human olfaction.
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Characterization of Phenylpropene O-Methyltransferases from Sweet Basil: Facile Change of Substrate Specificity and Convergent Evolution within a Plant O-Methyltransferase Family
The Plant Cell, 2002Co-Authors: David R Gang, Noa Lavid, Chloe Zubieta, Joseph P. Noel, Till Beuerle, Efraim Lewinsohn, Feng Chen, Eran PicherskyAbstract:Some basil varieties are able to convert the phenylpropenes chavicol and eugenol to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs encode proteins that are 90% identical to each other and very similar to several isoflavone O-methyltransferases such as IOMT, which catalyzes the 4′-O-methylation of 2,7,4′-trihydroxyisoflavanone. On the other hand, CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia breweri, indicating that the eugenol O-methylating enzymes in basil and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were highly expressed in the peltate glandular trichomes on the surface of the young basil leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that a single amino acid difference was responsible for the difference in substrate discrimination between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis, in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced that exhibited the opposite substrate preference relative to the respective native enzyme.
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Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits
PLANT PHYSIOLOGY, 2001Co-Authors: Efraim Lewinsohn, Olga Larkov, Kyoung Hee Nam, Fernond Schalechet, Jack Wilkinson, Kenji Matsui, Yaakov Tadmor, Orit Amar, Elena Lastochkin, Uzi RavidAbstract:The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.
Jihong Wang - One of the best experts on this subject based on the ideXlab platform.
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Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.
Archives of Biochemistry and Biophysics, 1999Co-Authors: Jihong Wang, Eran PicherskyAbstract:Abstract Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl- l -methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl- l -methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.
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Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.
Archives of Biochemistry and Biophysics, 1999Co-Authors: Jihong Wang, Eran PicherskyAbstract:Abstract Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl- l -methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl- l -methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.
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Floral scent production in Clarkia breweri. III. Enzymatic synthesis and emission of benzenoid esters
Plant Physiology, 1998Co-Authors: Natalia Dudareva, Robert A Raguso, Jihong Wang, Jeannine R. Ross, Eran PicherskyAbstract:The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.
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Characterization of S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase involved in floral scent production in Clarkia breweri.
Archives of Biochemistry and Biophysics, 1998Co-Authors: Jihong Wang, Eran PicherskyAbstract:Abstract Eugenol, isoeugenol, methyleugenol, and isomethyleugenol are volatiles found in the floral scent ofClarkia breweri.With their distinct aromas, they are used in many perfumes and food seasonings. Here we report the purification and characterization of (iso)eugenolO-methyltransferase (IEMT), the enzyme that methylates eugenol or isoeugenol to make methyleugenol or isomethyleugenol, respectively, usingS-adenosyl- l -methionine as the methyl donor.C. breweriIEMT was copurified with caffeic acidO-methyltransferase (COMT) from petals and purified to homogeneity from a bacterial expression system. IEMT is active as a homodimer with a subunit molecular mass of 40 kDa. It is stable at temperatures up to 35°C. It shows optimum activity at pH 7.5, and it does not require any cofactors for enzymatic activity. Plant-purified IEMT hasKmvalues of 7 and 58 μM for eugenol and isoeugenol, respectively, and 27 μM for SAM (30, 74, and 19 μM, respectively, for the plant IEMT expressed inEscherichia coli). By substituting coding regions from COMT into IEMT, it was determined that the regions in IEMT involved in substrate specificity are located in the first half of the protein sequence and that a small segment of 82 amino acids (amino acids 92–173) accounts for the main differences between IEMT and COMT in both substrate specificity and methylation regiospecificity.
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floral scent production in Clarkia breweri onagraceae ii localization and developmental modulation of the enzyme s adenosyl l methionine iso eugenol o methyltransferase and phenylpropanoid emission
Plant Physiology, 1997Co-Authors: Jihong Wang, Robert A Raguso, Natalia Dudareva, Shyam Bhakta, Eran PicherskyAbstract:We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55-67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4'-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.
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Electroantennogram responses ofHyles lineata (Sphingidae: Lepidoptera) to volatile compounds fromClarkia breweri (Onagraceae) and other moth-pollinated flowers
Journal of Chemical Ecology, 1996Co-Authors: Robert A Raguso, Douglas M Light, Eran PickerskyAbstract:Electroantennograms (EAGs) from field-collected Hyles lineata moths were recorded in response to 10 individual floral volatiles identified from Clarkia breweri (Onagraceae), to 22 scent compounds produced by other moth-pollinated flowers and to eight ubiquitous “green leaf volatiles.” Females' EAGs were generally 1.5- to 2-fold greater than those observed for male moths. Female:male EAG rank orders were significantly correlated, but marked differences in order were observed for some compounds (e.g., benzyl alcohol, cinnamic aldehyde, geraniol, and linalool). Linalool, benzyl acetate, methyl salicylate, and pyranoid linalool oxide elicited the largest EAG responses (−1.2 to −0.8 mV) among scent compounds from C. breweri . EAG responses were significantly lower for monoterpenes as a pooled compound class than for aromatic esters, alcohols and aldehydes, fatty acid derivatives, N-bearing compounds and oxygenated terpenoids. EAG responses to structurally related scent compounds were not significantly different in most cases. Both male and female H. lineata were sensitive to most C. breweri scent compounds at 10^−2 to 10^−4 µg/µl doses, and rank order in potency varied with the dose/concentration tested. H. lineata's olfactory sensitivity to diverse volatile compounds across a range of doses/concentrations suggests that a broad array of volatiles could function as floral attractants for foraging hawkmoths.
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electroantennogram responses of hyles lineata sphingidae lepidoptera to volatile compounds from Clarkia breweri onagraceae and other moth pollinated flowers
Journal of Chemical Ecology, 1996Co-Authors: Robert A Raguso, Douglas M Light, Eran PickerskyAbstract:Electroantennograms (EAGs) from field-collectedHyles lineata moths were recorded in response to 10 individual floral volatiles identified fromClarkia breweri (Onagraceae), to 22 scent compounds produced by other moth-pollinated flowers and to eight ubiquitous “green leaf volatiles.” Females' EAGs were generally 1.5- to 2-fold greater than those observed for male moths. Female:male EAG rank orders were significantly correlated, but marked differences in order were observed for some compounds (e.g., benzyl alcohol, cinnamic aldehyde, geraniol, and linalool). Linalool, benzyl acetate, methyl salicylate, and pyranoid linalool oxide elicited the largest EAG responses (−1.2 to −0.8 mV) among scent compounds fromC. breweri. EAG responses were significantly lower for monoterpenes as a pooled compound class than for aromatic esters, alcohols and aldehydes, fatty acid derivatives, N-bearing compounds and oxygenated terpenoids. EAG responses to structurally related scent compounds were not significantly different in most cases. Both male and femaleH. lineata were sensitive to mostC. breweri scent compounds at 10−2 to 10−4 µg/µl doses, and rank order in potency varied with the dose/concentration tested.H. lineata's olfactory sensitivity to diverse volatile compounds across a range of doses/concentrations suggests that a broad array of volatiles could function as floral attractants for foraging hawkmoths.