Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

Metabolites in the kynurenine pathway, generated by tryptophan degradation, are thought to play an important role in neurodegenerative disorders, including Alzheimer's and Huntington's diseases. In these disorders, glutamate receptor-mediated excitotoxicity and free radical formation have been correlated with decreased levels of the neuroprotective metabolite kynurenic acid. Here, we describe the synthesis and characterization of JM6, a small-molecule prodrug inhibitor of kynurenine 3-monooxygenase (KMO). Chronic oral administration of JM6 inhibits KMO in the blood, increasing kynurenic acid levels and reducing extracellular glutamate in the brain. In a transgenic mouse model of Alzheimer's disease, JM6 prevents spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extends life span, prevents synaptic loss, and decreases microglial activation in a mouse model of Huntington's disease. These findings support a critical link between tryptophan metabolism in the blood and neurodegeneration, and they provide a foundation for treatment of neurodegenerative diseases.     

Monitoring fibril formation of the N-terminal domain of PABPN1 carrying an alanine repeat by tryptophan fluorescence and real-time NMR

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.     

Energetics and Surface Properties of Pseudomonas putida DOT-T1E in a Two-Phase Fermentation System with 1-Decanol as Second Phase

The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl₂. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.     

Genome evolution in a Mediterranean species complex: phylogeny and cytogenetics of Helictotrichon (Poaceae) allopolyploids based on nuclear DNA sequences (rDNA,topoisomerase gene) and FISH

We examined the molecular phylogeny and chromosomal features of European Helictotrichon species to explore the relationships within the genus and to investigate the origin of several polyploids. Using both approaches, molecular and cytogenetic, revealed the strong impact of allopolyploidization on genome organization from chromosome structure to sequence level. Our research focused on Mediterranean and endemic species of the Alps. Altogether, the molecular phylogenetic analyses include a sample of 17 Helictotrichon species and subspecies, used DNA sequences from the nuclear ribosomal (nr) internal transcribed spacer region (ITS) and the single copy gene topoisomerase 6 (Topo6), and were analysed by maximum parsimony and Bayesian methods. Karyotype structures were investigated by fluorescence in situ hybridization (FISH) and fluorochrome banding. Cytogenetic characters were mapped on the combined phylogenetic tree. The absence or comparatively rare occurrence of different ITS sequence types in some (allo-) polyploid species of Helictotrichon suggests frequent intergenomic homogenization of ribosomal DNA (rDNA) loci due to the phenomenon of concerted evolution. This result implies that the ITS region is not an ideal marker to study polyploid evolution of these grasses. The phylogenetic analysis of the Topo6 region revealed three major clades that concur with three different copy types (termed SAR, SET, PAR), representing the major genome groups in Helictotrichon. A comparison of the molecular phylogenetic trees with the chromosome and karyotype structure supports allopolyploidy of several Helictotrichon species and identifies potential genome donors. A correlation between molecular phylogenetic/cytogenetic results and geographic distribution is expressed by a west-east disjunction, in the narrower or wider sense, of the analysed species. While SAR represents a geographically narrowly distributed southwest Mediterranean genome group, PAR and SET are very widespread (Mediterranean to Asia) and encompass several instances of west-east disjunctions.     

Integrated approach for production of recombinant acetylacetone dioxygenase from Acinetobacter johnsonii

The C–C bond-cleaving acetylacetone dioxygenase Dke1 (EC 1.13.11.50) is a Fe^2?+?-dependent enzyme from Acinetobacter johnsonii that activates oxygen to convert a range of β-dicarbonyl substrates into α-oxo-aldehyde and acid products. Previous methods of downstream processing yielded Dke1 with substoichiometric Fe^2?+? content. This paper reports the integration of enzyme production in E. coli and affinity chromatography to prepare recombinant Dke1 that is completely loaded with its metal cofactor. The specific activity of Dke1 in E. coli cell extracts could be increased up to 20-fold, compared to optimized enzyme production with the natural host. Introduction of an affinity-tag allowed the isolation of fully active Dke1 in a single purification step with high yield (70%). Mass spectrometric analysis revealed at the level of >80% sequence coverage that the isolated enzyme corresponded exactly to the predicted gene product. Tagged Dke1 is shown to have retained the functional properties of native Dke1.     

Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.     

Chiral Hydroxylation at the Mononuclear Nonheme Fe(II) Center of 4-(S) Hydroxymandelate Synthase – A Structure-Activity Relationship Analysis

(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.     

Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors

1. Download high-res image (143KB)
2. Download full-size image