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Publications - Molecular Signal Processing

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Publications

Kopycki, J.; Schmidt, J.; Abel, S.; Grubb, C. D.; Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus Biotechnol. Lett. 33, 1039-1046, (2011) DOI: 10.1007/s10529-011-0530-y

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.
Publications

Ludwig-Müller, J.; Denk, K.; Cohen, J. D.; Quint, M.; An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis J. Plant Growth Regul. 29, 242-248, (2010) DOI: 10.1007/s00344-009-9128-1

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).
Publications

Ziegler, J.; Facchini, P. J.; Geißler, R.; Schmidt, J.; Ammer, C.; Kramell, R.; Voigtländer, S.; Gesell, A.; Pienkny, S.; Brandt, W.; Evolution of morphine biosynthesis in opium poppy Phytochemistry 70, 1696-1707, (2009) DOI: 10.1016/j.phytochem.2009.07.006

Benzylisoquinoline alkaloids (BIAs) are a group of nitrogen-containing plant secondary metabolites comprised of an estimated 2500 identified structures. In BIA metabolism, (S)-reticuline is a key branch-point intermediate that can be directed into several alkaloid subtypes with different structural skeleton configurations. The morphinan alkaloids are one subclass of BIAs produced in only a few plant species, most notably and abundantly in the opium poppy (Papaver somniferum). Comparative transcriptome analysis of opium poppy and several other Papaver species that do not accumulate morphinan alkaloids showed that known genes encoding BIA biosynthetic enzymes are expressed at higher levels in P. somniferum. Three unknown cDNAs that are co-ordinately expressed with several BIA biosynthetic genes were identified as enzymes in the pathway. One of these enzymes, salutaridine reductase (SalR), which is specific for the production of morphinan alkaloids, was isolated and heterologously overexpressed in its active form not only from P. somniferum, but also from Papaver species that do not produce morphinan alkaloids. SalR is a member of a class of short chain dehydrogenase/reductases (SDRs) that are active as monomers and possess an extended amino acid sequence compared with classical SDRs. Homology modelling and substrate docking revealed the substrate binding site for SalR. The amino acids residues conferring salutaridine binding were compared to several members of the SDR family from different plant species, which non-specifically reduce (−)-menthone to (+)-neomenthol. Previously, it was shown that some of these proteins are involved in plant defence. The recruitment of specific monomeric SDRs from monomeric SDRs involved in plant defence is discussed.
Publications

Pienkny, S.; Brandt, W.; Schmidt, J.; Kramell, R.; Ziegler, J.; Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L) Plant J. 60, 56-67, (2009) DOI: 10.1111/j.1365-313X.2009.03937.x

The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species‐specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full‐length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O ‐methyltransferase protein family. It was related to O ‐methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6‐O ‐methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K m of 44 μm using S ‐adenosyl‐l ‐methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7–O ‐methyltransferase (N7OMT). This enzyme represents a novel O ‐methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.
Publications

Lee, C.-W.; Efetova, M.; Engelmann, J. C.; Kramell, R.; Wasternack, C.; Ludwig-Müller, J.; Hedrich, R.; Deeken, R.; Agrobacterium tumefaciens Promotes Tumor Induction by Modulating Pathogen Defense in Arabidopsis thaliana Plant Cell 21, 2948-2962, (2009) DOI: 10.1105/tpc.108.064576

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.
Publications

Jindaprasert, A.; Springob, K.; Schmidt, J.; De-Eknamkul, W.; Kutchan, T. M.; Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum Phytochemistry 69, 3043-3053, (2008) DOI: 10.1016/j.phytochem.2008.03.013

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51–67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.
Publications

Fellenberg, C.; Milkowski, C.; Hause, B.; Lange, P.-R.; Böttcher, C.; Schmidt, J.; Vogt, T.; Tapetum-specific location of a cation-dependent O-methyltransferase in Arabidopsis thaliana Plant J. 56, 132-145, (2008) DOI: 10.1111/j.1365-313X.2008.03576.x

Cation‐ and S ‐adenosyl‐l ‐methionine (AdoMet)‐dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT‐like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi‐suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N  ′,N  ′′‐ bis‐(5‐hydroxyferuloyl)‐N  ′′′‐sinapoylspermidine compensated for by N1 ,N5 ,N10 ‐tris‐(5‐hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5‐hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi‐mediated suppression data of the corresponding gene suggest a role of this cation‐dependent CCoAOMT‐like protein in the stamen/pollen development of A. thaliana .
Publications

Ziegler, J.; Voigtländer, S.; Schmidt, J.; Kramell, R.; Miersch, O.; Ammer, C.; Gesell, A.; Kutchan, T. M.; Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis Plant J. 48, 177-192, (2006) DOI: 10.1111/j.1365-313X.2006.02860.x

Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.
Publications

Meixner, C.; Ludwig-Müller, J.; Miersch, O.; Gresshoff, P.; Staehelin, C.; Vierheilig, H.; Lack of mycorrhizal autoregulation and phytohormonal changes in the supernodulating soybean mutant nts1007 Planta 222, 709-715, (2005) DOI: 10.1007/s00425-005-0003-4

Autoregulatory mechanisms have been reported in the rhizobial and the mycorrhizal symbiosis. Autoregulation means that already existing nodules or an existing root colonization by an arbuscular mycorrhizal fungus systemically suppress subsequent nodule formation/root colonization in other parts of the root system. Mutants of some legumes lost their ability to autoregulate the nodule number and thus display a supernodulating phenotype. On studying the effect of pre-inoculation of one side of a split-root system with an arbuscular mycorrhizal fungus on subsequent mycorrhization in the second side of the split-root system of a wild-type soybean (Glycine max L.) cv. Bragg and its supernodulating mutant nts1007, we observed a clear suppressional effect in the wild-type, whereas further root colonization in the split-root system of the mutant nts1007 was not suppressed. These data strongly indicate that the mechanisms involved in supernodulation also affect mycorrhization and support the hypothesis that the autoregulation in the rhizobial and the mycorrhizal symbiosis is controlled in a similar manner. The accumulation patterns of the plant hormones IAA, ABA and Jasmonic acid (JA) in non-inoculated control plants and split-root systems of inoculated plants with one mycorrhizal side of the split-root system and one non-mycorrhizal side, indicate an involvement of IAA in the autoregulation of mycorrhization. Mycorrhizal colonization of soybeans also resulted in a strong induction of ABA and JA levels, but on the basis of our data the role of these two phytohormones in mycorrhizal autoregulation is questionable.
Publications

Grubb, C. D.; Zipp, B. J.; Ludwig-Müller, J.; Masuno, M. N.; Molinski, T. F.; Abel, S.; Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis Plant J. 40, 893-908, (2004) DOI: 10.1111/j.1365-313X.2004.02261.x

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.
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