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Publications - Cell and Metabolic Biology

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Publications

Hans, J.; Brandt, W.; Vogt, T.; Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis Plant J. 39, 319-333, (2004) DOI: 10.1111/j.1365-313X.2004.02133.x

In livingstone daisy (Dorotheanthus bellidiformis ), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.
Publications

Walter, M. H.; Hans, J.; Strack, D.; Two distantly related genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots Plant J. 31, 243-254, (2002) DOI: 10.1046/j.1365-313X.2002.01352.x

Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1‐deoxy‐d ‐xylulose 5‐phosphate synthase (DXS), which was previously thought to be encoded by a single‐copy gene. We have identified two distinct classes of DXS‐like cDNAs from the model legume Medicago truncatula . The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid‐targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula , maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene‐synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.
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