Structural and molecular genetic insight into a widespread sulfur oxidation pathway.

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a DeltadsrE mutant experiment that the cytoplasmic alpha2beta2gamma2-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 A resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.

Allochromatium vinosum DsrC: solution-state NMR structure, redox properties, and interaction with DsrEFH, a protein essential for purple sulfur bacterial sulfur oxidation.

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein, as well as with an electron-transfer function via thiol-disulfide interchanges.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DsrEFH from Allochromatium vinosum.

In purple sulfur bacteria, the proteins encoded by dsr genes play an essential role in the oxidation of intracellular sulfur, which is an obligate intermediate during the oxidation of sulfide and thiosulfate. One such gene product, DsrEFH from Allochromatium vinosum, has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.5 A from a crystal of selenomethionine-substituted DsrEFH. The crystal belongs to the primitive monoclinic space group P2(1), with unit-cell parameters a = 56.6, b = 183.1, c = 107.8 A, beta = 99.6 degrees. A full structure determination is under way in order to provide insight into the structure-function relationships of this protein.

Novel genes of the dsr gene cluster and evidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum.

Seven new genes designated dsrLJOPNSR were identified immediately downstream of dsrABEFHCMK, completing the dsr gene cluster of the phototrophic sulfur bacterium Allochromatium vinosum D (DSM 180(T)). Interposon mutagenesis proved an essential role of the encoded proteins for the oxidation of intracellular sulfur, an obligate intermediate during the oxidation of sulfide and thiosulfate. While dsrR and dsrS encode cytoplasmic proteins of unknown function, the other genes encode a predicted NADPH:acceptor oxidoreductase (DsrL), a triheme c-type cytochrome (DsrJ), a periplasmic iron-sulfur protein (DsrO), and an integral membrane protein (DsrP). DsrN resembles cobyrinic acid a,c-diamide synthases and is probably involved in the biosynthesis of siro(heme)amide, the prosthetic group of the dsrAB-encoded sulfite reductase. The presence of most predicted Dsr proteins in A. vinosum was verified by Western blot analysis. With the exception of the constitutively present DsrC, the formation of Dsr gene products was greatly enhanced by sulfide. DsrEFH were purified from the soluble fraction and constitute a soluble alpha(2)beta(2)gamma(2)-structured 75-kDa holoprotein. DsrKJO were purified from membranes pointing at the presence of a transmembrane electron-transporting complex consisting of DsrKMJOP. In accordance with the suggestion that related complexes from dissimilatory sulfate reducers transfer electrons to sulfite reductase, the A. vinosum Dsr complex is copurified with sulfite reductase, DsrEFH, and DsrC. We therefore now have an ideal and unique possibility to study the interaction of sulfite reductase with other proteins and to clarify the long-standing problem of electron transport from and to sulfite reductase, not only in phototrophic bacteria but also in sulfate-reducing prokaryotes.

Cerebrospinal fluid levels of beta-amyloid(42) in patients with Alzheimer's disease are related to the exon 2 polymorphism of the cathepsin D gene.

The intracellular aspartyl protease cathepsin D (catD) is involved in such Alzheimer's disease (AD)-related processes as the activation of the endosomal/lysosomal system and the cleavage of the amyloid precursor protein into amyloidogenic components, which may initiate neurodegeneration. A non-synonymous polymorphism (exon 2, C to T exchange leading to ala-->val substitution) of the gene encoding catD (CTSD) was previously associated with AD, in that the T allele increased the risk for AD. To investigate whether the T allele is associated with disease-related traits, we measured the concentration of the amyloid beta-peptide 1-42 (Abeta(42)) and 1-40 (Abeta(40)) in patients and control subjects. The T allele of the CTSD genotype was associated with a 50% decrease in Abeta(42) levels in the cerebrospinal fluid. Thus, we demonstrate a significant impact of the CTSD genotype on Abeta(42) levels in the cerebrospinal fluid of AD patients and underpin the importance of the validation of susceptibility genes by examining their potential pathophysiological relevance.