Discovery of a novel transcriptional regulator of sugar catabolism in archaea.

The haloarchaeon Haloferax volcanii degrades D-glucose via the semiphosphorylative Entner-Doudoroff pathway and D-fructose via a modified Embden-Meyerhof pathway. Here, we report the identification of GfcR, a novel type of transcriptional regulator that functions as an activator of both D-glucose and D-fructose catabolism. We find that in the presence of D-glucose, GfcR activates gluconate dehydratase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase and also acts as activator of the phosphotransferase system and of fructose-1,6-bisphosphate aldolase, which are involved in uptake and degradation of D-fructose. In addition, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase are activated by GfcR in the presence of D-fructose and also during growth on D-galactose and glycerol. Electrophoretic mobility shift assays indicate that GfcR binds directly to promoters of regulated genes. Specific intermediates of the degradation pathways of the three hexoses and of glycerol were identified as inducer molecules of GfcR. GfcR is composed of a phosphoribosyltransferase (PRT) domain with an N-terminal helix-turn-helix motif and thus shows homology to PurR of Gram-positive bacteria that is involved in the transcriptional regulation of nucleotide biosynthesis. We propose that GfcR of H. volcanii evolved from a PRT-like enzyme to attain a function as a transcriptional regulator of central sugar catabolic pathways in archaea.

Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase From the Hyperthermophilic Archaeon Thermofilum sp.

A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4-1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.

Pentose degradation in archaea: Halorhabdus species degrade D-xylose, L-arabinose and D-ribose via bacterial-type pathways.

The degradation of the pentoses D-xylose, L-arabinose and D-ribose in the domain of archaea, in Haloferax volcanii and in Haloarcula and Sulfolobus species, has been shown to proceed via oxidative pathways to generate α-ketoglutarate. Here, we report that the haloarchaeal Halorhabdus species utilize the bacterial-type non-oxidative degradation pathways for pentoses generating xylulose-5-phosphate. The genes of these pathways are each clustered and were constitutively expressed. Selected enzymes involved in D-xylose degradation, xylose isomerase and xylulokinase, and those involved in L-arabinose degradation, arabinose isomerase and ribulokinase, were characterized. Further, D-ribose degradation in Halorhabdus species involves ribokinase, ribose-5-phosphate isomerase and D-ribulose-5-phosphate-3-epimerase. Ribokinase of Halorhabdus tiamatea and ribose-5-phosphate isomerase of Halorhabdus utahensis were characterized. This is the first report of pentose degradation via the bacterial-type pathways in archaea, in Halorhabdus species that likely acquired these pathways from bacteria. The utilization of bacterial-type pathways of pentose degradation rather than the archaeal oxidative pathways generating α-ketoglutarate might be explained by an incomplete gluconeogenesis in Halorhabdus species preventing the utilization of α-ketoglutarate in the anabolism.

D-galactose catabolism in archaea: operation of the DeLey-Doudoroff pathway in Haloferax volcanii.

The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey-Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey-Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey-Doudoroff pathway in the domain of archaea.

d-Ribose Catabolism in Archaea: Discovery of a Novel Oxidative Pathway in Haloarcula Species.

The Haloarcula species H. marismortui and H. hispanica were found to grow on d-ribose, d-xylose, and l-arabinose. Here, we report the discovery of a novel promiscuous oxidative pathway of pentose degradation based on genome analysis, identification and characterization of enzymes, transcriptional analysis, and growth experiments with knockout mutants. Together, the data indicate that in Haloarcula spp., d-ribose, d-xylose, and l-arabinose were degraded to α-ketoglutarate involving the following enzymes: (i) a promiscuous pentose dehydrogenase that catalyzed the oxidation of d-ribose, d-xylose, and l-arabinose; (ii) a promiscuous pentonolactonase that was involved in the hydrolysis of ribonolactone, xylonolactone, and arabinolactone; (iii) a highly specific dehydratase, ribonate dehydratase, which catalyzed the dehydration of ribonate, and a second enzyme, a promiscuous xylonate/gluconate dehydratase, which was involved in the conversion of xylonate, arabinonate, and gluconate. Phylogenetic analysis indicated that the highly specific ribonate dehydratase constitutes a novel sugar acid dehydratase family within the enolase superfamily; and (iv) finally, 2-keto-3-deoxypentanonate dehydratase and α-ketoglutarate semialdehyde dehydrogenase catalyzed the conversion of 2-keto-3-deoxypentanonate to α-ketoglutarate via α-ketoglutarate semialdehyde. We conclude that the expanded substrate specificities of the pentose dehydrogenase and pentonolactonase toward d-ribose and ribonolactone, respectively, and the presence of a highly specific ribonate dehydratase are prerequisites of the oxidative degradation of d-ribose in Haloarcula spp. This is the first characterization of an oxidative degradation pathway of d-ribose to α-ketoglutarate in archaea.IMPORTANCE The utilization and degradation of d-ribose in archaea, the third domain of life, have not been analyzed so far. We show that Haloarcula species utilize d-ribose, which is degraded to α-ketoglutarate via a novel oxidative pathway. Evidence is presented that the oxidative degradation of d-ribose involves novel promiscuous enzymes, pentose dehydrogenase and pentonolactonase, and a novel sugar acid dehydratase highly specific for ribonate. This is the first report of an oxidative degradation pathway of d-ribose in archaea, which differs from the canonical nonoxidative pathway of d-ribose degradation reported for most bacteria. The data contribute to our understanding of the unusual sugar degradation pathways and enzymes in archaea.

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases.

Pyruvate kinases (PKs) synthesize ATP as the final step of glycolysis in the three domains of life. PKs from most bacteria and eukarya are allosteric enzymes that are activated by sugar phosphates; for example, the feed-forward regulator fructose-1,6-bisphosphate, or AMP as a sensor of energy charge. Archaea utilize unusual glycolytic pathways, but the allosteric properties of PKs from these species are largely unknown. Here, we present an analysis of 24 PKs from most archaeal clades with respect to allosteric properties, together with phylogenetic analyses constructed using a novel mode of rooting protein trees. We find that PKs from many Thermoproteales, an order of crenarchaeota, are allosterically activated by 3-phosphoglycerate (3PG). We also identify five conserved amino acids that form the binding pocket for 3PG. 3PG is generated via an irreversible reaction in the modified glycolytic pathway of these archaea and therefore functions as a feed-forward regulator. We also show that PKs from hyperthermophilic Methanococcales, an order of euryarchaeota, are activated by AMP. Phylogenetic analyses indicate that 3PG-activated PKs form an evolutionary lineage that is distinct from that of sugar-phosphate activated PKs, and that sugar phosphate-activated PKs originated as AMP-regulated PKs in hyperthermophilic Methanococcales. Since the phospho group of sugar phosphates and 3PG overlap in the allosteric site, our data indicate that the allostery in PKs first started from a progenitor phosphate-binding site that evolved in two spatially distinct directions: one direction generated the canonical site that responds to sugar phosphates and the other gave rise to the 3PG site present in Thermoproteales. Overall, our data suggest an intimate connection between the allosteric properties and evolution of PKs.

l-Rhamnose catabolism in archaea.

The halophilic archaeon Haloferax volcanii utilizes l-rhamnose as a sole carbon and energy source. It is shown that l-rhamnose is taken up by an ABC transporter and is oxidatively degraded to pyruvate and l-lactate via the diketo-hydrolase pathway. The genes involved in l-rhamnose uptake and degradation form a l-rhamnose catabolism (rhc) gene cluster. The rhc cluster also contains a gene, rhcR, that encodes the transcriptional regulator RhcR which was characterized as an activator of all rhc genes. 2-keto-3-deoxy-l-rhamnonate, a metabolic intermediate of l-rhamnose degradation, was identified as inducer molecule of RhcR. The essential function of rhc genes for uptake and degradation of l-rhamnose was proven by the respective knockout mutants. Enzymes of the diketo-hydrolase pathway, including l-rhamnose dehydrogenase, l-rhamnonolactonase, l-rhamnonate dehydratase, 2-keto-3-deoxy-l-rhamnonate dehydrogenase and 2,4-diketo-3-deoxy-l-rhamnonate hydrolase, were characterized. Further, genes of the diketo-hydrolase pathway were also identified in the hyperthermophilic crenarchaeota Vulcanisaeta distributa and Sulfolobus solfataricus and selected enzymes were characterized, indicating the presence of the diketo-hydrolase pathway in these archaea. Together, this is the first comprehensive description of l-rhamnose catabolism in the domain of archaea.

Facial attractiveness: visual impact of symmetry increases significantly towards the midline.

Symmetry is thought to be a major prerequisite for an attractive face. Many faces are not symmetric, yet are still regarded as beautiful. What role, then, does asymmetry play in the perception of beauty? We studied the assessment of computer-manipulated images by independent judges (n = 200-250): part A: nevi located at different positions; part B: standardized changes of the orbital region. The results showed that slight lateral orbital and facial asymmetry does not impair attractiveness at all and that asymmetries close to the midline are significantly less attractive than those affecting the lateral aspect of the face (P < 0.001). A single nevus which is located laterally on the face is significantly more attractive than a nevus close to the midline (P < 0.001). Faces with a completely symmetric bilateral pair of nevi in the same lateral positions (perceived as attractive when alone), received the worst ratings (P < 0.001). Symmetry is a characteristic of the attractive face, but there are exceptions to the rule. Under certain conditions symmetry can be completely unattractive. The visual impact of symmetry on the perception of beauty increases significantly when approaching the midline.

[Suspicious circumstances on discovery of the cadaver]. / Suspekte Leichenauffindesituationen.

The authors report on three autopsy cases in which the findings at the death scene gave rise to the suspect of non-natural, in two cases even violent causes of death. The medico-legal and criminalistic investigations led to surprising results: Death was due to acute intoxication with narcotics, supposed delirium tremens and a suicide attempt in the presence of bronchopneumonia. External force was ruled out in all three fatalities by cooperative work of police investigators and forensic medicine.