Interconverting the catalytic activities of (betaalpha)(8)-barrel enzymes from different metabolic pathways: sequence requirements and molecular analysis

The (betaalpha)(8)-barrel enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (tHisA) and imidazole glycerol phosphate synthase (tHisF) from Thermotoga maritima catalyze two successive reactions in the biosynthesis of histidine. In both enzymes, aspartate residues at the C-terminal end of beta-strand 1 (Asp8 in tHisA and Asp11 in tHisF) and beta-strand 5 (Asp127 in tHisA and Asp130 in tHisF) are essential for catalytic activity. It was demonstrated earlier that in tHisA the substitution of Asp127 by valine (tHisA-D127V) generates phosphoribosylanthranilate isomerase (TrpF) activity, a related (betaalpha)(8)-barrel enzyme participating in tryptophan biosynthesis. It is shown here that in tHisF the corresponding substitution of Asp130 by valine (tHisF-D130V) also generates TrpF activity. To determine the effectiveness of individual amino acid exchanges in these conversions, each of the 20 standard amino acid residues was introduced at position 127 of tHisA and 130 of tHisF by saturation random mutagenesis. The tHisA-D127X and tHisF-D130X variants with TrpF activity were identified by selection in vivo, and the proteins purified and characterized. The results obtained show that removal of the negatively charged carboxylate side-chain at the C-terminal end of beta-strand 5 is sufficient to establish TrpF activity in tHisA and tHisF, presumably because it allows the binding of the negatively charged TrpF substrate, phosphoribosylanthranilate. In contrast, the double mutants tHisA-D8N+D127V and tHisF-D11N+D130V did not show detectable activity, demonstrating that the aspartate residues at the C-terminal end of beta-strand 1 are essential for catalysis of the TrpF reaction. The ease with which TrpF activity can be established on both the tHisA and tHisF scaffolds supports the evolutionary relationship of these three enzymes and highlights the functional plasticity of the (betaalpha)(8)-barrel enzyme fold.     

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.     

The Shark HoxN Cluster Is Homologous to the Human HoxD Cluster

The statistical analysis of phylogenetic footprints in the two known horn shark Hox clusters and the four mammalian clusters shows that the shark HoxN cluster is HoxD-like. This finding implies that the most recent common ancestor of jawed vertebrates had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters.     

A comparison of the intestinal helminth communities of Equidae in Southern Africa

The intestinal helminth communities of 8 horses, 12 donkeys, 21 Hartmann's mountain zebras, and 44 Burchell's zebras were compared using the original data from 6 studies in South Africa and Namibia. Necropsy and worm recovery techniques were comparable between the studies. Sixty helminth species (58 nematode, 1 cestode, and 1 trematode species) were recorded. There were significant differences in the helminth community structures of the 4 Equus species. The helminth communities of the 2 closely related zebra subspecies were most similar, and they jointly shared 7 helminth species with donkeys and only 1 with horses. Geographic variation and host-mixing contributed to the helminth species composition. Multiple confamilial species infections were the norm in the donkeys and zebra subspecies, and no single-species infection was recorded for the Strongylidae. Congeneric species were commonly recorded in 3 genera (Cyathostomum, Cylicocyclus, and Cylicostephanus). The shape of the occupancy frequency distributions for the donkeys and zebra subspecies was multimodal, with no clear satellite or core modes. Despite the presence of environmental variability and comparatively low parasite-host specificity, the phylogenetic signal within Equus helminth communities remains strong.     

The accumulation of alpha-zein in transgenic tobacco endosperm is stabilized by co-expression of beta-zein

The cysteine-poor alpha-zein is the major prolamin storage protein fraction in maize endosperm and is localized in the interior of protein bodies with delta-zein, whereas the hydrophobic cysteine-rich beta- and gamma-zein are found on the exterior of the PB. In transgenic tobacco endosperm expressing zein genes, alpha-zein was unstable unless co-expressed with gamma-zein. Here we showed that alpha-zein was also stabilized by beta-zein. Small accretions of alpha- and beta-zeins, similar in appearance to maize protein bodies, were localized to the endoplasmic reticulum within tobacco endosperm cells. The zein proteins were also localized to protein storage vacuoles in a more dispersed pattern, suggesting that they were transported there after they were post-translationally sequestered into the ER.     

Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I

This study describes the discovery of a new inherited disorder of glycosylation named "CDG-Ik." CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I [MIM 605907]), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis. Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy. One patient had a homozygous point mutation (c.773C-->T, S258L), whereas the other patient was compound heterozygous for the mutations c.773C-->T and c.1025A-->C (E342P). Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles.     

Sphingosine kinase 1 is an intracellular effector of phosphatidic acid

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). Because both substrate and product of the enzyme are potentially important signaling molecules, the regulation of SK1 is of considerable interest. We report that SK1, which is ordinarily a cytosolic enzyme, translocates in vivo and in vitro to membrane compartments enriched in phosphatidic acid (PA), the lipid product of phospholipase D. This translocation depends on direct interaction of SK1 with PA, because recombinant purified enzyme shows strong affinity for pure PA coupled to Affi-Gel. The SK1-PA interaction maps to the C terminus of SK1 and is independent of catalytic activity or of the diacylglycerol kinase-like domain of the enzyme. Thus SK1 constitutes a novel, physiologically relevant PA effector.     

Genome size in Echinops L. and related genera (Asteraceae, Cardueae): karyological, ecological and phylogenetic implications

Genome size was assessed by flow cytometry in 33 species belonging to seven genera of the tribe Cardueae (Asteraceae), which can be grouped in three taxonomic complexes. 2C nuclear DNA content ranged from 1.49 to 16.98 pg, which is more than elevenfold variation. Genome size correlated well with some karyological traits. Nuclear DNA amount variations also have systematic and evolutionary implications and/or are linked to adaptations to ecological conditions.