No abstract

YdiB and its paralog AroE are members of the quinate/ shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out sitedirected mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents.

No abstract

Most familial breast and ovarian cancers have been linked to mutations in the BRCA1 gene. BRCA1 has been shown to affect gene transcription but how it does so remains elusive. Here we show that BRCA1 can stimulate transcription without the requirement for a DNA-tethering function in mammalian and yeast cells. Furthermore, the BRCA1 C-terminal region can stimulate transcription of the p53-responsive promoter, MDM2. Unlike many enhancer-specific activators, non-tethered BRCA1 does not require a functional TATA element to stimulate transcription. Our results suggest that BRCA1 can enhance transcription by a function additional to recruiting the transcriptional machinery to a targeted gene.

No abstract