The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The fumarate sensor DcuS is a prototype for a two component sensory histidine kinase with signal perception in the periplasm, transmembrane signal transfer (1, 2), and autophosphorylation of a His residue in the kinase domain in the cytoplasm (3). DcuS belongs to the CitA family of sensors that are specific for sensing di-and tricarboxylates (1, 2, 4, 5). The periplasmic domain of the histidine autokinase CitA works as a highly specific citrate receptor, whereas DcuS uses any type of C 4 -dicarboxylate, like fumarate, succinate, and malate, as a stimulus (1, 4 -6). DcuS is predicted to consist of two transmenbrane helices and of a periplasmic sensory domain enclosed by the transmembrane helices. The second transmembrane helix is followed by a cytoplasmic PAS 1 domain of unknown function and the kinase with the consensus histidine residue for autophosphorylation. The periplasmic citrate binding domain of CitA is conserved in DcuS and presumably responsible for binding of fumarate and other C 4 -dicarboxylates. Preliminary results suggest that fumarate sensing occurs by this domain in the periplasm (2, 4, 5). After phosphorylation by DcuS the response regulator DcuR of the DcuSR system activates the expression of the target genes like dcuB and frd-ABCD encoding an anaerobic fumarate carrier DcuB and fumarate reductase (4, 5). Despite their prevalence no structural information is available for transmembranous sensory kinases, in particular not for signal perception and transmission across the membrane. Only the structures of cytoplasmic sensory kinases, or of domains not involved in transmembrane signaling, have been determined.Purified DcuS is active after reconstitution in proteoliposomes and capable of transmembranous stimulation of the kinase by fumarate (2). For a more detailed understanding of signal perception representing the first step of signal transduction in transmembranous histidine kinases of two-component systems, the structure of the periplasmic C 4 -dicarboxylate binding domain of DcuS (DcuS-pd) was determined after stable over-production of the domain. ("DcuS-pd")-The sequence of dcuS coding for the periplasmic domain of DcuS (DcuS or DcuS-pd) enclosed by the two transmembrane helices was cloned into the NdeI and HindIII sites of plasmid pET28a (Novagen) resulting in plasmid pMW145. The DNA fragment was amplified with oligonucleotides pdcus-NdeII (ATT TAC TTC TCG CAT ATG AGT GAT ATG) and pdcuSHind (GAC CAG ATA AAG CTT CAG CGA CTG) by PCR of genomic Escheric...

Sagittamide A is a long-chain acyclic α,ω-dicarboxylic acid with eight stereocenters. The central hexahydroxyhexane moiety carries six of them. For this moiety, two different relative stereochemical assignments were published in 2006. In this communication, one relative configuration is clearly singled out based on NOEs, J- and residual dipolar couplings despite conformational averaging that is present in this moiety. The proposed NMR method relies on the measurement of dipolar couplings in recently introduced alignment media that are compatible with organic solvents, and cross validating the NMR determined ensemble of conformations against the residual dipolar couplings. The method is easily applicable to many other stereochemical problems.

The computer program COCON is introduced as a tool for the comprehensive structure elucidation of unknown organic compounds. In particular, structural proposals made on the basis of the molecular formula and of 2D‐NMR experiments can be analyzed for the existence of alternative constitutions being in agreement with the same data set. The computational speed grounds on the evaluation of ambiguous long‐range connectivity information during the process of structure generation. The data set experimentally obtained for the marine natural product oroidin (1) was selected, because proton‐poor compounds usually cause uncertainties in NMR‐based structure determinations. The calculation results encourage to move from the experience‐based analysis of NMR chemical shifts or of MS fragmentations to the automated evaluation of routinely available connectivity information.