Twists and turns: a tale of two shikimate-pathway enzymes

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Difluorinated analogues of shikimic acid

Investigations into the quinate to shikimate transformation have been carried out, the results of which have been exploited in the synthesis of a novel difluoromethylene homologue of shikimic acid from (-)-quinic acid. Martin's sulfurane {Ph2S[OC(CF3)(2)Ph](2)} was the reagent of choice for the key dehydration step of this synthesis. The results of investigations into the synthesis of the important natural product analogue, 6,6-difluoroshikimic acid are also reported. (C) 2003 Elsevier Science Ltd. All rights reserved.

A molecular and structural characterization of senescing Arabidopsis siliques and comparison of transcriptional profiles with senescing petals and leaves

Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling, and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis thaliana, the seeds are contained within a silique, which is itself a photosynthetic organ in the early stages of development and undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins, and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analysis software (MapMan). In all three tissues, members of NAC and WRKY transcription factor families were up-regulated, but components of the shikimate and cell-wall biosynthetic pathways were down-regulated during senescence. Expression of genes encoding ethylene biosynthesis and action showed more similarity between senescing siliques and petals than between senescing siliques and leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilization phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and discussed.