Publications by authors named "Mohammed Saddik Motawia"

45 Publications

An independent evolutionary origin for insect deterrent cucurbitacins in Iberis amara.

Mol Biol Evol 2021 Jul 15. Epub 2021 Jul 15.

Department of Plant and Environmental Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

Pieris rapae and Phyllotreta nemorum are Brassicaceae specialists, but do not feed on Iberis amara spp. that contain cucurbitacins. The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Using de-novo assembled transcriptomics from I. amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase (IaCPQ). In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyse the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the draft genome assembly of I. amara showed that the IaCPQ was localized to the same scaffold together with CYP708A15 but spanning over 100 kb, this contrasts with the highly organized cucurbitacin gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This study thus provides new insight into the mechanism of recurrent evolution and diversification of a plant defensive chemical.
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http://dx.doi.org/10.1093/molbev/msab213DOI Listing
July 2021

A flavin-dependent monooxygenase catalyzes the initial step in cyanogenic glycoside synthesis in ferns.

Commun Biol 2020 09 11;3(1):507. Epub 2020 Sep 11.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis-the conversion of an amino acid to the corresponding aldoxime-is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.
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http://dx.doi.org/10.1038/s42003-020-01224-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486406PMC
September 2020

Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in .

J Biol Chem 2020 07 3;295(31):10581-10592. Epub 2020 Jun 3.

Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.
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http://dx.doi.org/10.1074/jbc.RA120.013878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397118PMC
July 2020

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Metabolites during Sorghum Germination.

Plant Physiol 2020 07 29;183(3):925-942. Epub 2020 Apr 29.

VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg 1871, Denmark

Dhurrin is the most abundant cyanogenic glucoside found in sorghum ( where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination.
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http://dx.doi.org/10.1104/pp.19.01357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333723PMC
July 2020

Phenolic cross-links: building and de-constructing the plant cell wall.

Nat Prod Rep 2020 07 23;37(7):919-961. Epub 2020 Jan 23.

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2 generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.
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http://dx.doi.org/10.1039/c9np00028cDOI Listing
July 2020

Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent.

Phytochemistry 2020 Feb 30;170:112214. Epub 2019 Nov 30.

Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark; Center for Synthetic Biology "bioSYNergy", Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark; VILLUM Research Center "Plant Plasticity", Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark. Electronic address:

In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate environment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes.
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http://dx.doi.org/10.1016/j.phytochem.2019.112214DOI Listing
February 2020

An Optimized Screen Reduces the Number of GA Transporters and Provides Insights Into Nitrate Transporter 1/Peptide Transporter Family Substrate Determinants.

Front Plant Sci 2019 3;10:1106. Epub 2019 Oct 3.

DynaMo Center, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.

Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes, wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities toward organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis suggests distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.
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http://dx.doi.org/10.3389/fpls.2019.01106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785635PMC
October 2019

Defining optimal electron transfer partners for light-driven cytochrome P450 reactions.

Metab Eng 2019 09 12;55:33-43. Epub 2019 May 12.

Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. Electronic address:

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.
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http://dx.doi.org/10.1016/j.ymben.2019.05.003DOI Listing
September 2019

Deletion of biosynthetic genes, specific SNP patterns and differences in transcript accumulation cause variation in hydroxynitrile glucoside content in barley cultivars.

Sci Rep 2019 04 5;9(1):5730. Epub 2019 Apr 5.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Copenhagen, Denmark.

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides, potentially involved in alleviating pathogen and environmental stresses. These compounds include the cyanogenic glucoside epiheterodendrin. The biosynthetic genes are clustered. Total hydroxynitrile glucoside contents were previously shown to vary from zero to more than 10,000 nmoles g in different barley lines. To elucidate the cause of this variation, the biosynthetic genes from the high-level producer cv. Mentor, the medium-level producer cv. Pallas, and the zero-level producer cv. Emir were investigated. In cv. Emir, a major deletion in the genome spanning most of the hydroxynitrile glucoside biosynthetic gene cluster was identified and explains the complete absence of hydroxynitrile glucosides in this cultivar. The transcript levels of the biosynthetic genes were significantly higher in the high-level producer cv. Mentor compared to the medium-level producer cv. Pallas, indicating transcriptional regulation as a contributor to the variation in hydroxynitrile glucoside levels. A correlation between distinct single nucleotide polymorphism (SNP) patterns in the biosynthetic gene cluster and the hydroxynitrile glucoside levels in 227 barley lines was identified. It is remarkable that in spite of the demonstrated presence of a multitude of SNPs and differences in transcript levels, the ratio between the five hydroxynitrile glucosides is maintained across all the analysed barley lines. This implies the involvement of a stably assembled multienzyme complex.
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http://dx.doi.org/10.1038/s41598-019-41884-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450869PMC
April 2019

Vitamin D in Arabidopsis thaliana.

Sci Rep 2018 11 5;8(1):16348. Epub 2018 Nov 5.

Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, F-67083, Strasbourg, France.

Vitamin D is a secosterol hormone critical for bone growth and calcium homeostasis, produced in vertebrate skin by photolytic conversion of the cholesterol biosynthetic intermediate provitamin D. Insufficient levels of vitamin D especially in the case of low solar UV-B irradiation is often compensated by an intake of a dietary source of vitamin D of animal origin. Small amounts of vitamin D were described in a few plant species and considered as a peculiar feature of their phytochemical diversity. In this report we show the presence of vitamin D in the model plant Arabidopsis thaliana. This plant secosterol is a UV-B mediated derivative of provitamin D, the precursor of sitosterol. The present work will allow a further survey of vitamin D distribution in plant species.
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http://dx.doi.org/10.1038/s41598-018-34775-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218535PMC
November 2018

Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum.

Plant J 2018 06 19;94(6):1109-1125. Epub 2018 May 19.

VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, 1871, Denmark.

Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.
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http://dx.doi.org/10.1111/tpj.13923DOI Listing
June 2018

Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes.

J Exp Bot 2017 Nov;68(20):5527-5537

Section for Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark.

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.
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http://dx.doi.org/10.1093/jxb/erx362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853437PMC
November 2017

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity.

Sci Rep 2017 08 24;7(1):9326. Epub 2017 Aug 24.

School of Agriculture, Food and Rural Development, Newcastle University, NE1 7RU, Newcastle upon Tyne, UK.

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.
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http://dx.doi.org/10.1038/s41598-017-04307-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570955PMC
August 2017

Total biosynthesis of the cyclic AMP booster forskolin from .

Elife 2017 03 14;6. Epub 2017 Mar 14.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant , in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13-manoyl oxide into forskolin as demonstrated by transient expression in . The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into and afforded forskolin titers of 40 mg/L.
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http://dx.doi.org/10.7554/eLife.23001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388535PMC
March 2017

Origin and evolution of transporter substrate specificity within the NPF family.

Elife 2017 03 3;6. Epub 2017 Mar 3.

DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark.

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the . Biochemical characterization of orthologs along the phylogenetic lineage from cassava to suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.
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http://dx.doi.org/10.7554/eLife.19466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336358PMC
March 2017

The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling.

Phytochemistry 2017 Mar 11;135:8-17. Epub 2017 Jan 11.

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark; Center for Synthetic Biology bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark. Electronic address:

The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s.
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http://dx.doi.org/10.1016/j.phytochem.2016.11.013DOI Listing
March 2017

Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum.

Science 2016 11;354(6314):890-893

Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.

Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.
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http://dx.doi.org/10.1126/science.aag2347DOI Listing
November 2016

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

Sci Rep 2016 11 14;6:37079. Epub 2016 Nov 14.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark.

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.
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http://dx.doi.org/10.1038/srep37079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107947PMC
November 2016

High throughput screening of starch structures using carbohydrate microarrays.

Sci Rep 2016 07 29;6:30551. Epub 2016 Jul 29.

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches.
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http://dx.doi.org/10.1038/srep30551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965820PMC
July 2016

Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis.

ACS Chem Biol 2016 07 4;11(7):1862-9. Epub 2016 May 4.

Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.
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http://dx.doi.org/10.1021/acschembio.6b00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949584PMC
July 2016

Lotus japonicus flowers are defended by a cyanogenic β-glucosidase with highly restricted expression to essential reproductive organs.

Plant Mol Biol 2015 Sep 7;89(1-2):21-34. Epub 2015 Aug 7.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 1871, Frederiksberg C, Denmark.

Flowers and leaves of Lotus japonicus contain α-, β-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by β-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.
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http://dx.doi.org/10.1007/s11103-015-0348-4DOI Listing
September 2015

Glucosinolate-related glucosides in Alliaria petiolata: sources of variation in the plant and different metabolism in an adapted specialist herbivore, Pieris rapae.

J Chem Ecol 2014 Oct 12;40(10):1063-79. Epub 2014 Oct 12.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.

Specialized metabolites in plants influence their interactions with other species, including herbivorous insects, which may adapt to tolerate defensive phytochemicals. The chemical arsenal of Alliaria petiolata (garlic mustard, Brassicaceae) includes the glucosinolate sinigrin and alliarinoside, a hydroxynitrile glucoside with defensive properties to glucosinolate-adapted specialists. To further our understanding of the chemical ecology of A. petiolata, which is spreading invasively in North America, we investigated the metabolite profile and here report a novel natural product, petiolatamide, which is structurally related to sinigrin. In an extensive study of North American populations of A. petiolata, we demonstrate that genetic population differences as well as developmental regulation contribute to variation in the leaf content of petiolatamide, alliarinoside, sinigrin, and a related glycoside. We furthermore demonstrate widely different metabolic fates of these metabolites after ingestion in the glucosinolate-adapted herbivore Pieris rapae, ranging from simple passage over metabolic conversion to sequestration. The differences in metabolic fate were influenced by plant β-glucosidases, insect-mediated degradation, and the specificity of the larval gut transport system mediating sequestration.
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http://dx.doi.org/10.1007/s10886-014-0509-yDOI Listing
October 2014

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

Nat Commun 2014 Jun 19;5:4037. Epub 2014 Jun 19.

1] Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [2] VILLUM Research Center 'Plant Plasticity', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [3] Center for Synthetic Biology: 'bioSYNergy', Thorvaldsensvej 40, Frederiksberg C, DK-1871 Copenhagen, Denmark [4] Carlsberg Laboratory, Gamle Carlsberg Vej 10, Valby DK-2500, Copenhagen, Denmark.

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.
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http://dx.doi.org/10.1038/ncomms5037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083428PMC
June 2014

Synthesis of the allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America.

Carbohydr Res 2014 Jul 17;394:13-6. Epub 2014 May 17.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; VILLUM Research Center for 'Plant Plasticity', University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; Center for Synthetic Biology 'bioSYNergy', University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. Electronic address:

The allelochemical alliarinoside present in garlic mustard (Alliaria petiolata), an invasive plant species in North America, was chemically synthesized using an efficient and practical synthetic strategy based on a simple reaction sequence. Commercially available 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose was converted into prop-2-enyl 2',3',4',6'-tetra-O-acetyl-β-D-glucopyranoside and subjected to epoxidation. In a one-pot reaction, ring-opening of the epoxide using TMSCN under solvent free conditions followed by treatment of the formed trimethylsilyloxy nitrile with pyridine and phosphoryl chloride, afforded the acetylated β-unsaturated nitriles (Z)-4-(2',3',4',6'-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile and its isomer (E)-4-(2',3',4',6'-tetra-O-β-D-glucopyranosyloxy)but-2-enenitrile. Deacetylation of Z- and/or E-isomers afforded the target molecules alliarinoside and its isomer.
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http://dx.doi.org/10.1016/j.carres.2014.05.006DOI Listing
July 2014

Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore.

Insect Biochem Mol Biol 2014 Jan 21;44:44-53. Epub 2013 Nov 21.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; Villum research center "Plant Plasticity" University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.

Considering the staggering diversity of bioactive natural products present in plants, insects are only able to sequester a small number of phytochemicals from their food plants. The mechanisms of how only some phytochemicals are sequestered and how the sequestration process takes place remains largely unknown. In this study the model system of Zygaena filipendulae (Lepidoptera) and their food plant Lotus corniculatus is used to advance the knowledge of insect sequestration. Z. filipendulae larvae are dependent on sequestration of the cyanogenic glucosides linamarin and lotaustralin from their food plant, and have a much lower fitness if reared on plants without these compounds. This study investigates the fate of the cyanogenic glucosides during ingestion, sequestration in the larvae, and in the course of insect ontogeny. To this purpose, double-labeled linamarin and lotaustralin were chemically synthesized carrying two stable isotopes, a (2)H labeled aglucone and a (13)C labeled glucose moiety. In addition, a small amount of (14)C was incorporated into the glucose residue. The isotope-labeled compounds were applied onto cyanogenic L. corniculatus leaves that were subsequently presented to the Z. filipendulae larvae. Following ingestion by the larvae, the destiny of the isotope labeled cyanogenic glucosides was monitored in different tissues of larvae and adults at selected time points, using radio-TLC and LC-MS analyses. It was shown that sequestered compounds are taken up intact, contrary to earlier hypotheses where it was suggested that the compounds would have to be hydrolyzed before transport across the gut. The uptake from the larval gut was highly stereo selective as the β-glucosides were retained while the α-glucosides were excreted and recovered in the frass. Sequestered compounds were rapidly distributed into all analyzed tissues of the larval body, partly retained throughout metamorphosis and transferred into the adult insect where they were distributed to all tissues. During subsequent mating, isotope labeled cyanogenic glucosides were transferred from the male to the female in the nuptial gift.
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http://dx.doi.org/10.1016/j.ibmb.2013.11.003DOI Listing
January 2014

The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography-mass spectrometry based metabolomics.

Anal Bioanal Chem 2013 Nov 4;405(28):9193-205. Epub 2013 Oct 4.

Quality and Technology, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, Frederiksberg C, 1958, Copenhagen, Denmark,

Reproducible and quantitative gas chromatography-mass spectrometry (GC-MS)-based metabolomics analysis of complex biological mixtures requires robust and broad-spectrum derivatization. We have evaluated derivatization of complex metabolite mixtures using trimethylsilyl cyanide (TMSCN) and the most commonly used silylation reagent N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). For the comparative analysis, two metabolite mixtures, a standard complex mixture of 35 metabolites covering a range of amino acids, carbohydrates, small organic acids, phenolic acids, flavonoids and triterpenoids, and a phenolic extract of blueberry fruits were used. Four different derivatization methods, (1) direct silylation using TMSCN, (2) methoximation followed by TMSCN (M-TMSCN), (3) direct silylation using MSTFA, and (4) methoximation followed by MSTFA (M-MSTFA) were compared in terms of method sensitivity, repeatability, and derivatization reaction time. The derivatization methods were observed at 13 different derivatization times, 5 min to 60 h, for both metabolite mixtures. Fully automated sample derivatization and injection enabled excellent repeatability and precise method comparisons. At the optimal silylation times, peak intensities of 34 out of 35 metabolites of the standard mixture were up to five times higher using M-TMSCN compared with M-MSTFA. For direct silylation of the complex standard mixture, the TMSCN method was up to 54 times more sensitive than MSTFA. Similarly, all the metabolites detected from the blueberry extract showed up to 8.8 times higher intensities when derivatized using TMSCN than with MSTFA. Moreover, TMSCN-based silylation showed fewer artifact peaks, robust profiles, and higher reaction speed as compared with MSTFA. A method repeatability test revealed the following robustness of the four methods: TMSCN > M-TMSCN > M-MSTFA > MSTFA.
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http://dx.doi.org/10.1007/s00216-013-7341-zDOI Listing
November 2013

Male-to-female transfer of 5-hydroxytryptophan glucoside during mating in Zygaena filipendulae (Lepidoptera).

Insect Biochem Mol Biol 2013 Nov 3;43(11):1037-44. Epub 2013 Sep 3.

Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; Villum Research Center "Plant Plasticity", Denmark. Electronic address:

Zygaena filipendulae accumulates the cyanogenic glucosides linamarin and lotaustralin by larval sequestration from the food plant or de novo biosynthesis. We have previously demonstrated that the Z. filipendulae male transfers linamarin and lotaustralin to the female in the course of mating. In this study we report the additional transfer of 5-hydroxytryptophan glucoside (5-(β-d-glucopyranosyloxy)-L-Tryptophan) from the Z. filipendulae male internal genitalia to the female spermatophore around 5 h into the mating process. 5-Hydroxytryptophan glucoside is present in the virgin male internal genitalia, and production continues during the early phase of mating. Following initiation of 5-hydroxytryptophan glucoside transfer to the female, the amount in male internal genitalia is drastically reduced until after mating where it is slowly replenished. For unambiguous structural identification, 5-hydroxytryptophan glucoside was chemically synthesized and used as an authentic standard. The biological function of 5-hydroxytryptophan glucoside remains to be established, although we have indications that it may be involved in inducing the female to stay in copula and delay egg-laying to prevent re-mating of the female. To our knowledge 5-hydroxytryptophan glucoside has not previously been reported present in animal tissues.
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http://dx.doi.org/10.1016/j.ibmb.2013.08.007DOI Listing
November 2013

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants.

Proc Natl Acad Sci U S A 2013 Aug 12;110(35):E3360-7. Epub 2013 Aug 12.

Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom.

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family--as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.
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http://dx.doi.org/10.1073/pnas.1309157110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3761579PMC
August 2013

Biosynthesis of rhodiocyanosides in Lotus japonicus: rhodiocyanoside A is synthesized from (Z)-2-methylbutanaloxime via 2-methyl-2-butenenitrile.

Phytochemistry 2012 May 3;77:260-7. Epub 2012 Mar 3.

Plant Biochemistry Laboratory, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark.

Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound.
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http://dx.doi.org/10.1016/j.phytochem.2012.01.020DOI Listing
May 2012
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