Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of 18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.IS

Degradation of haloaromatic compounds

An ever increasing number of halogenated organic compounds has been produced by industry in the last few decades. These compounds are employed as biocides, for synthetic polymers, as solvents, and as synthetic intermediates. Production figures are often incomplete, and total production has frequently to be extrapolated from estimates for individual countries. Compounds of this type as a rule are highly persistent against biodegradation and belong, as "recalcitrant" chemicals, to the class of so-called xenobiotics. This term is used to characterise chemical substances which have no or limited structural analogy to natural compounds for which degradation pathways have evolved over billions of years. Xenobiotics frequently have some common features. e.g. high octanol/water partitioning coefficients and low water solubility which makes for a high accumulation ratio in the biosphere (bioaccumulation potential). Recalcitrant compounds therefore are found accumulated in mammals, especially in fat tissue, animal milk supplies and also in human milk. Highly sophisticated analytical techniques have been developed for the detection of organochlorines at the trace and ultratrace level

Systemic Gene Expression in Arabidopsis during an Incompatible Interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of “high alert,” otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the β-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks

Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory

Application of the [gamma-P-32] ATP kinase assay to study anabolic signaling in human skeletal muscle

AMPK (AMP-dependant protein kinase)-mTORC1 (mechanistic target of rapamycin in complex 1)-p70S6K1 (ribosomal protein S6 kinase 1 of 70 kDa) signaling plays a crucial role in muscle protein synthesis (MPS). Understanding this pathway has been advanced by the application of the Western blot (WB) technique. However, because many components of the mTORC1 pathway undergo numerous, multisite posttranslational modifications, solely studying the phosphorylation changes of mTORC1 and its substrates may not adequately represent the true metabolic signaling processes. The aim of this study was to develop and apply a quantitative in vitro [γ-32P] ATP kinase assay (KA) for p70S6K1 to assess kinase activity in human skeletal muscle to resistance exercise (RE) and protein feeding. In an initial series of experiments the assay was validated in tissue culture and in p70S6K1-knockout tissues. Following these experiments, the methodology was applied to assess p70S6K1 signaling responses to a physiologically relevant stimulus. Six men performed unilateral RE followed by the consumption of 20 g of protein. Muscle biopsies were obtained at pre-RE, and 1 and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity as assessed by the KA was significantly increased from pre-RE at 1 and 3 h post-RE. However, phosphorylated p70S6K1thr389 was not significantly elevated. AMPK activity was suppressed from pre-RE at 3 h post-RE, whereas phosphorylated ACCser79 was unchanged. Total protein kinase B activity also was unchanged after RE from pre-RE levels. Of the other markers we assessed by WB, 4EBP1thr37/46 phosphorylation was the only significant responder, being elevated at 3 h post-RE from pre-RE. These data highlight the utility of the KA to study skeletal muscle plasticity