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.
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.
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 Km of 44 lM 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 Omethyltransferase 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.
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.
Fellenberg, C.; Milkowski, C.; Hause, B.; Lange, P.; 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.
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 22.214.171.124). 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.
Gidda, K.S.; Miersch, O.; Schmidt, J.; Wasternack, C.; Varin, L. Biochemical and molecular characterization of a hydroxy-jasmonate sulfotransferase from Arabidopsis thaliana J. Biol. Chem. 278, 17895-17900, (2003) DOI: 10.1074/jbc.M211943200
12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with Km values of 50 and 10 µM, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring in A. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.
Books and chapters
Bachmann, A.; Kohlmann, M.; Wasternack, C.; Feussner, I. Oxylipins in sorbitol-stressed barley leaf segments (Sánchez, J., Cerdá-Olmedo, E., Martínez-Force, E.). Secretariado de Publicaciones de la Universidad de Sevilla 288-290, (1998)
Miersch, O.; Knöfel, H.-D.; Schmidt, J.; Kramell, R.; Parthier, B. A jasmonic acid conjugate, N-[()-jasmonoyl]-tyramine, from Petunia pollen Phytochemistry 47, 327-329, (1998)
Kramell, R.; Atzorn, R.; Schneider, G.; Miersch, O.; Brückner, C.; Schmidt, J.; Sembdner, G.; Parthier, B. Occurrence and identification of jasmonic acid and its amino acid conjugates induced by osmotic stress in barley leaf tissue J. Plant Growth Reg. 14, 29-36, (1995)