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Publikationen - Molekulare Signalverarbeitung

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Publikation

Ziegler, J.; Brandt, W.; Geißler, R.; Facchini, P. J. Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis J Biol Chem 284, 26758-26767, (2009) DOI: 10.1074/jbc.M109.030957

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel -helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking alsorevealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalRwas created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.
Publikation

Parry, G.; Calderón Villalobos, L.I.; Prigge, M.; Peret, B.; Dharmasiri, S.; Itoh, H.; Lechner, E.; Gray, W.M.; Bennett, M.; Estelle, M. Complex regulation of the TIR/AFB family of auxin receptors Proc Natl Acad Sci USA 106(52), 22540-22545, (2009) DOI: 10.1073/pnas.0911967106

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.
Publikation

Mugford, S.G.; Yoshimoto, N.; Reichelt, M.; Wirtz, M.; Hill, L.; Mugford, S.T.; Nakazato, Y.; Noji, M.; Takahashi, H.; Kramell, R.; Gigolashvili, T.; Flügge, U.-I.; Wasternack, C.; Gershenzon, J.; Hell, R.; Saito, K.; Kopriva, S. Disruption of Adenosine-5'-Phosphosulfate Kinase in <span>Arabidopsis</span> Reduces Levels of Sulfated Secondary Metabolites Plant Cell 21, 910-927, (2009) DOI: 10.1105/tpc.109.065581

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 59-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 39-phosphoadenosine 59-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normalgrowth rates.
Publikation

Quint, M.; Barkawi, L.S.; Fan, K.T.; Cohen, J.D.; Gray, W.M. Arabidopsis IAR4 modulates auxin response by regulating auxin homeostasis Plant Physiol 150, 748-758, (2009) DOI: 10.1104/pp.109.136671

In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mitochondrial pyruvate dehydrogenase E1α-subunit, IAA-Alanine Resistant4 (IAR4). In addition to enhancing the auxin response defects of tir1-1, iar4 single mutants exhibit numerous auxin-related phenotypes including auxin-resistant root growth and reduced lateral root development, as well as defects in primary root growth, root hair initiation, and root hair elongation. Remarkably, all of these iar4 mutant phenotypes were rescued when endogenous indole-3-acetic acid (IAA) levels were increased by growth at high temperature or overexpression of the YUCCA1 IAA biosynthetic enzyme, suggesting that iar4 mutations may alter IAA homeostasis rather than auxin response. Consistent with this possibility, iar4 mutants exhibit increased Aux/IAA stability compared to wild type under basal conditions, but not in response to an auxin treatment. Measurements of free IAA levels detected no significant difference between iar4-3 and wild-type controls. However, we consistently observed significantly higher levels of IAA-amino acid conjugates in the iar4-3 mutant. Furthermore, using stable isotope-labeled IAA precursors, we observed a significant increase in the relative utilization of the Trp-independent IAA biosynthetic pathway in iar4-3. We therefore suggest that the auxin phenotypes of iar4 mutants are the result of altered IAA homeostasis.
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