Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.)

Landmann, Christian; Hücherig, Stefanie; Fink, Barbara; Hoffmann, Thomas; Dittlein, Daniela; Coiner, Heather A.; Schwab, Wilfried

doi:10.1007/s00425-011-1400-5

Cited by 41 publications

(46 citation statements)

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“…S6). In addition, the ASAT1 enzyme showed evidence of substrate promiscuity as was previously documented for other BAHD enzymes (32). The aromatic benzoyl-CoA was efficiently used as a donor substrate with sucrose, whereas the negatively charged malonyl-CoA did not yield detectable product (SI Appendix, Table S3 and Fig.…”

Section: Sl-asat2supporting

confidence: 62%

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Fan

Miller

Schilmiller

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

114

233

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Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used-in concert with two previously identified BAHD acyltransferases-to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates.Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.Solanum | glandular trichomes | acylsugar | specialized metabolism | genotype to phenotype P lants are masters of metabolism, producing hundreds of thousands of small molecules known as specialized metabolites, which vary widely in structure, abundance, and physical and biological properties. These metabolites tend to be produced by enzymes that evolve faster than those that produce "central" metabolites such as amino acids, nucleotides, sugars, and cofactors (1-3), and the pathways and metabolic intermediates involved in biosynthesis of many specialized metabolites remain mysterious. Despite the growing availability of genomic DNA sequences, understanding the genetic and biochemical mechanisms that contribute to this phenotypic diversity and plasticity presents enduring and major challenges in plant biochemistry. It is of great interest to understand and manipulate the biosynthesis of these biologically active molecules.Specialized metabolites typically are produced in a cell-or tissue-specific manner and are generally limited in their taxonomic distribution. Glandular secreting trichomes provide an example of such a differentiated structure; these epidermal "hairs" produce a variety of metabolites of importance to humans, including aromatic flavor components (e.g., in hops for beer and Mediterranean herbs for cooking), psychoactive cannabinoids in Cannabis, and the antimalarial drug artemisinin in Artemisia annua (4, 5).Some trichome-produced metabolites have documented direct and indirect antiherbivore activities (4, 6-8). For example, acylsugars are a group of structurally related specialized metabolites produced in plants of the nightshade family-the Solanaceae (9, 10). Characterized examples ...

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Section: Sl-asat2supporting

confidence: 62%

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Fan

Miller

Schilmiller

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

114

233

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“…Given that linalool is a tertiary alcohol (other monoterpenes studied here are primary or secondary alcohols), it is possible that the linalool acetyltransferase is different in structure and mechanism of action than enzymes reported here (Aharoni et al 2000;Beekwilder et al 2004;Zaks et al 2008). The optimum pH and temperature for LiAAT-3 and LiAAT-4 were in the range of alcohol acetyltransferases reported previously (Landmann et al 2011;Sharma et al 2013) and were determined to be 8.0-8.5 and 30-32°C, respectively. Both enzymes demonstrated linear catalytic activity from 10 to 120 min.…”

Section: Discussionmentioning

confidence: 99%

“…Transcriptional activity of transcripts in flower and glandular trichome tissues were normalized against leaf of their respective plants (Phylogenetic tree analysis of BAHD acyltransferases including LiAAT-1-4. The tree was constructed by neighbor-joining and bootstrap distance analysis (adapted from D'Auria 2006;Landmann et al 2011). Candidates included that have been characterized by either genetic mutant screening or biochemical assay.…”

mentioning

confidence: 99%

Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of L. x intermedia.

Sarker

Mahmoud

2015

Planta

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Two alcohol acetyltransferases, LiAAT-3 and LiAAT-4, from L. x intermedia were cloned, expressed in bacteria, and functionally characterized. Two monoterpene acetyltransferase cDNA clones (LiAAT-3 and LiAAT-4) were isolated from L. x intermedia glandular trichomes, expressed in bacteria to produce, and functionally characterize the encoded proteins in vitro. The recombinant LiAAT-3 and LiAAT-4 proteins had molecular weights of ca. 47 and 49 kDa, respectively, as evidenced by SDS-PAGE. The K m (mM) values for the recombinant LiAAT-3 and LiAAT-4 were 1.046 and 0.354 for lavandulol, 1.31 and 0.279 for geraniol, and 0.87 and 0.113 for nerol, respectively. The V max (pkat/mg) values for LiAAT-3 and LiAAT-4 were 92.13 and 105.1 for lavandulol, 81.07 and 52.17 for geraniol, and 15.02 and 15.8 for nerol, correspondingly. Catalytic efficiencies (mM(-1) min(-1)) for LiAAT-3 and LiAAT-4 were 0.27 and 0.85 for lavandulol, 0.19 and 0.54 for geraniol, and 0.052 and 0.4 for nerol, respectively. These kinetic properties are in the range of those reported for other plant acetyltransferases, and indicate that LiAAT-4 has a better catalytic efficiency than LiAAT-3, with lavandulol serving as the preferred substrate for both enzymes. Transcripts for both genes were abundant in L. angustifolia and L. x intermedia flowers, where monoterpene acetates are produced, and were undetectable (or present in trace quantities) in L. latifolia flowers, which do not accumulate significant amounts of these metabolites.

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“…Each of these specific mechanisms is prevalent in specialized metabolic enzymes. Many enzyme classes are well known for showing high levels of substrate permissiveness, especially those involved in tailoring reactions that decorate and modulate the physiochemical features of core chemical scaffolds, for example, oxidative hydroxylation (Ohnishi et al 2012), methylation (Kopycki et al 2008;Huang et al 2012), acylation (Landmann et al 2011), glycosidation (Lim et al 2001), adenylation (Schneider et al 2005;Westfall et al 2012), prenylation (Kumano et al 2010), and redox reactions (Kim et al 2004;Huang et al 2009). These enzymes often act on structurally related substrates (Fig.…”

Section: The Role Of Catalytic Promiscuity In Enzyme Evolvabilitymentioning

confidence: 99%

“…For example, the plant BAHD acyltransferases often recognize a variety of acyl acceptors, producing multiple esters and amides using the same acyl donor molecule (Landmann et al 2011). Similarly, the plant GH3 acyl acid amido synthetase family conjugates various amino acids onto the same acyl donor (Fig.…”

Section: The Role Of Catalytic Promiscuity In Enzyme Evolvabilitymentioning

confidence: 99%

The Remarkable Pliability and Promiscuity of Specialized Metabolism

Weng

Noel

2012

Cold Spring Harbor Symposia on Quantitative Biology

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Metabolic pathways are often considered "perfected" or at least predictable as substrates efficiently rearrange into products through the intervention of an optimized enzyme. Moreover, single catalytic steps link up, forming a myriad of metabolic circuits that are often modeled with a high degree of certainty. However, on closer examination, most enzymes are not precise with respect to their activity, using not just one substrate but often a variety and producing not just one product but a diversity. Hence, the metabolic systems assembled from enzymes possessing varying degrees of what can be termed catalytic promiscuity are not clear-cut and restrictive; rather, they may at times operate stochastically in the intracellular milieu. This "messiness" complicates our understanding of normal and aberrant cellular behavior, while paradoxically sowing the seeds for future advantageous metabolic adaptations for host organisms. Catalytic promiscuity is intrinsically associated with the dynamic nature of enzyme structures and their chemical mechanisms, both key to enzyme and metabolic evolvability. In addition to primary (core) metabolism, which is essential for survival, organisms also possess highly elaborated secondary (specialized) metabolic systems. These specialized enzymes and pathways often provide unique adaptive strategies for a myriad of organisms and their populations in challenging and changing ecosystems. Generally, enzymes of specialized metabolism show attenuated kinetic activities and expanded catalytic promiscuity compared with their phylogenetic relatives rooted in primary metabolism. We propose that evolvability may be a selected trait in many specialized metabolic systems spread across populations of organisms exposed to continually fluctuating biotic and abiotic environmental pressures. As minor metabolites arising from catalytic messiness provided enhanced population fitness, specificity relaxed, and catalytic efficiency was attenuated. This updated view provides a mechanistic basis for reaching a deeper understanding of the evolutionary underpinnings of the explosion of chemodiversity in nature.Fundamentally, metabolism is a chemical property of living organisms, defined as the collection of enzymecatalyzed chemical reactions that synthesize or deconstruct metabolic chemicals necessary to sustain life and contribute to the reproductive success of host populations in the face of ever-changing environmental pressures. Similarly to other catalysts, enzymes do not alter the equilibrium of the chemical reactions but enhance the rate of the reactions by lowering their activation energies (Cook and Cleland 2007). Reactions catalyzed by enzymes are generally precise and efficient, involving specific metabolic substrates transformed into predictable metabolic products using defined catalytic mechanisms. This ordered view of one enzyme -one mechanism -one product comes from seminal discoveries made by many investigators during the last century, focused for excellent practical reasons on the phylogenetically...

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Substrate promiscuity of a rosmarinic acid synthase from lavender (Lavandula angustifolia L.)

Cited by 41 publications

References 44 publications

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of L. x intermedia.

The Remarkable Pliability and Promiscuity of Specialized Metabolism

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