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Publikationen - Molekulare Signalverarbeitung

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Publikation

Kopycki, J.; Wieduwild, E.; Kohlschmidt, J.; Brandt, W.; Stepanova, A.N.; Alonso, J.M.; Pedras, M.S.; Abel, S.; Grubb, C.D. Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition Biochem J 450, 37-46, (2013) DOI: 10.1042/BJ20121403

Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.
Publikation

Sharma, V.K.; Monostori, T.; Hause, B.; Maucher, H.; Göbel, C.; Hornung, E.; Hänsch, R.; Bittner, F.; Wasternack, C.; Feussner, I.; Mendel, R.R.; Schulze, J. Genetic transformation of barley to modify expression of a 13-lipoxygenase Acta Biol. Szeged 49, 33-34 , (2005)

Immature scutella of barley were transformed with cDNA coding for a 13-li-poxygenase of barley (LOX-100) via particle bombardment. Regenerated plants were tested by PAT-assay, Western-analysis and PCR-screening. Immunocytochemical assay of T0 plants showed expression of the LOX cDNA both in the chloroplasts and in the cytosol, depending on the presence of the chloroplast signal peptide sequences in the cDNA. A few transgenic plants containing higher amounts of LOX-derived products have been found. These are the candidates for further analysis concerning pathogen resistance.
Publikation

Berger, S.; Weichert, H.; Porzel, A.; Wasternack, C.; Kühn, H.; Feussner, I. Enzymatic and non-enzymatic lipid peroxidation in leaf development Biochim. Biophys. Acta 1533, 266-276, (2001)

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Publikation

Ziegler, J.; Vogt, T.; Miersch, O.; Strack, D. Concentration of dilute protein solutions prior to sodium dodecylsulfate polyacrylamide gel electrophoresis Anal. Biochem 250, 257-260, (1997) DOI: 10.1006/abio.1997.2248

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Publikation

Abel, S.; Nguyen, M.D.; Theologis, A. The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana Journal of Biological Chemistry 270, 19093-19099, (1995)

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 μM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.
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