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Targeted metabolic phenotyping reveals absence of a regulatory response to gene knock-outs
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural results implicate that these mutated residues in the ComA protein may be play an important role in the formation a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.
We conclude that the (well-studied) regulatory machinery enables the yeast cell to globally adjust to different substrates as dictated by the environment; this adjustment is strongly reflected in the metabolite levels. However, the cell seemingly does not have the circuitry to respond to genetic perturbations. Thus, if a knockout in central metabolism cannot be compensated locally by an already expressed isoenzyme, it is likely to impair growth. doi:10.1016/j.nbt.2009.06.976
doi:10.1016/j.nbt.2009.06.975
4.6.16 4.6.15 Targeted metabolic phenotyping reveals absence of a regulatory response to gene knock-outs ∗
J.C. Ewald , T. Matt, N. Zamboni
Targeting the peptide deformylase of Salmonella enterica for virtual screening and structure based drug designing P. Somvanshi ∗ , P.K. Seth Bioinformatics Centre, Biotech Park, Sector-G, Jankipuram, Lucknow, India
Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
How does metabolism respond to enzyme deletions? Loss of function mutations are both a relevant evolutionary phenomenon as well as an important tool in biological research and metabolic engineering. Saccharomyces cerevisiae has a large fraction of nonessential genes, and many gene deletions — even in core regions of metabolism — have no apparent effect on growth and viability. This robustness can be due to enzyme dispensability/inactivity or compensation by the cell in response to the lesion. Other gene deletions lead to strong growth phenotypes, i.e. the cell fails to counteract the loss of function, also in cases where alternative pathways are identified in silico. The mechanism leading to the observed phenotypes can only be investigated with tools that provide a read-out of the state of metabolism at the resolution of single reactions. To systematically elucidate whether and how the metabolic network adapts to gene deletions in metabolic enzymes, we grew 35 S. cerevisiae single deletion mutants on three different carbon sources: glucose (fermentative), galactose (respiratory) and ethanol (gluconeogenic), and monitored their metabolism by metabolomics. To achieve a sufficient through-put we developed a metabolomics workflow which allows the parallel cultivation, quenching and sample processing of 48 batch cultures. During exponential growth we quantified 60 intracellular metabolites in central-carbon and amino acid metabolism by LC—MS/MS and GC—TOF and determined physiology and metabolic fluxes, resulting in 420 quantitative data sets. Growth on different substrates led to global reallocation of metabolite pools in central carbon metabolism, which was common in all mutants. In contrast, the mutations mostly led to local responses which were specific for the gene deletion. We could distinguish three cases: (i) truly silent mutations, (ii) mutations which resulted in a metabolome but not in a flux change, and (iii) readjustment of fluxes (i.e. slow growth or alternative pathway usage) which always coincided with global changes in metabolite pools. The most commonly observed effect was an accumulation of educt of the deleted reaction which propagated backward up to the closest, upstream irreversible reaction.
S366
www.elsevier.com/locate/nbt
Peptide deformylase (PDF) is a bacterial metalloenzyme responsible for cleaving the formyl group from nascent polypeptides, aiding in their maturation. It plays pivotal role in the survival of bacterial cells which is conserved in the eubacteria that is considered an attractive target for developing new antibacterial agents. The homology modeling was employed for generation of 3-D structure of PDF of Salmonella enterica and showed the 91% amino acid in allowed region of Ramachandran plot. PDF was used as target for a set of six inhibitors with substantial structural differences. Docking results show that the BB-3497, Actinonin and BBS-02 bind with high affinity to enzyme active site. Phylogeny of PDF in S. enterica shows homology with other strains of pathogenic bacteria. These data validate PDF as a novel target for the design of a new generation of antimycobacterial agents. doi:10.1016/j.nbt.2009.06.977
4.6.17 DNA binding studies of Chloridazon K.M. Mehd 1,∗ , K. Soheila 1 , R. Hamideh 2 , P. Hossein 3 1
Faculty of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Razi University, Kermanshah, Islamic Republic of Iran 2 Faculty of Chemistry, Razi University, Kermanshah, Islamic Republic of Iran 3 Department of Chemistry, Faculty of Science, Azad University of Ilam, Islamic Republic of Iran
Herbicides have been extensively applied in recent decades in agriculture throughout the world. Chloridazon (5-amino-4chloro-2-phenyl-3(2H)-pyridazinone) is an herbicide which is widely used in sugar beet crops. Therefore the investigation of genotoxic potential of Chloridazon via its interaction with DNA and evaluation of DNA damage is very important. In this study, the DNA binding properties in HEPES buffer (pH 7.2), has been monitored as a function of Chloridazon—DNA molar ratio, by spectrophotometry and spectopolarimetry. It is found that Chloridazon molecules perhaps inserts between base
ABSTRACTS
has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural results implicate that these mutated residues in the ComA protein may be play an important role in the formation a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.
We conclude that the (well-studied) regulatory machinery enables the yeast cell to globally adjust to different substrates as dictated by the environment; this adjustment is strongly reflected in the metabolite levels. However, the cell seemingly does not have the circuitry to respond to genetic perturbations. Thus, if a knockout in central metabolism cannot be compensated locally by an already expressed isoenzyme, it is likely to impair growth. doi:10.1016/j.nbt.2009.06.976
doi:10.1016/j.nbt.2009.06.975
4.6.16 4.6.15 Targeted metabolic phenotyping reveals absence of a regulatory response to gene knock-outs ∗
J.C. Ewald , T. Matt, N. Zamboni
Targeting the peptide deformylase of Salmonella enterica for virtual screening and structure based drug designing P. Somvanshi ∗ , P.K. Seth Bioinformatics Centre, Biotech Park, Sector-G, Jankipuram, Lucknow, India
Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
How does metabolism respond to enzyme deletions? Loss of function mutations are both a relevant evolutionary phenomenon as well as an important tool in biological research and metabolic engineering. Saccharomyces cerevisiae has a large fraction of nonessential genes, and many gene deletions — even in core regions of metabolism — have no apparent effect on growth and viability. This robustness can be due to enzyme dispensability/inactivity or compensation by the cell in response to the lesion. Other gene deletions lead to strong growth phenotypes, i.e. the cell fails to counteract the loss of function, also in cases where alternative pathways are identified in silico. The mechanism leading to the observed phenotypes can only be investigated with tools that provide a read-out of the state of metabolism at the resolution of single reactions. To systematically elucidate whether and how the metabolic network adapts to gene deletions in metabolic enzymes, we grew 35 S. cerevisiae single deletion mutants on three different carbon sources: glucose (fermentative), galactose (respiratory) and ethanol (gluconeogenic), and monitored their metabolism by metabolomics. To achieve a sufficient through-put we developed a metabolomics workflow which allows the parallel cultivation, quenching and sample processing of 48 batch cultures. During exponential growth we quantified 60 intracellular metabolites in central-carbon and amino acid metabolism by LC—MS/MS and GC—TOF and determined physiology and metabolic fluxes, resulting in 420 quantitative data sets. Growth on different substrates led to global reallocation of metabolite pools in central carbon metabolism, which was common in all mutants. In contrast, the mutations mostly led to local responses which were specific for the gene deletion. We could distinguish three cases: (i) truly silent mutations, (ii) mutations which resulted in a metabolome but not in a flux change, and (iii) readjustment of fluxes (i.e. slow growth or alternative pathway usage) which always coincided with global changes in metabolite pools. The most commonly observed effect was an accumulation of educt of the deleted reaction which propagated backward up to the closest, upstream irreversible reaction.
S366
www.elsevier.com/locate/nbt
Peptide deformylase (PDF) is a bacterial metalloenzyme responsible for cleaving the formyl group from nascent polypeptides, aiding in their maturation. It plays pivotal role in the survival of bacterial cells which is conserved in the eubacteria that is considered an attractive target for developing new antibacterial agents. The homology modeling was employed for generation of 3-D structure of PDF of Salmonella enterica and showed the 91% amino acid in allowed region of Ramachandran plot. PDF was used as target for a set of six inhibitors with substantial structural differences. Docking results show that the BB-3497, Actinonin and BBS-02 bind with high affinity to enzyme active site. Phylogeny of PDF in S. enterica shows homology with other strains of pathogenic bacteria. These data validate PDF as a novel target for the design of a new generation of antimycobacterial agents. doi:10.1016/j.nbt.2009.06.977
4.6.17 DNA binding studies of Chloridazon K.M. Mehd 1,∗ , K. Soheila 1 , R. Hamideh 2 , P. Hossein 3 1
Faculty of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Razi University, Kermanshah, Islamic Republic of Iran 2 Faculty of Chemistry, Razi University, Kermanshah, Islamic Republic of Iran 3 Department of Chemistry, Faculty of Science, Azad University of Ilam, Islamic Republic of Iran
Herbicides have been extensively applied in recent decades in agriculture throughout the world. Chloridazon (5-amino-4chloro-2-phenyl-3(2H)-pyridazinone) is an herbicide which is widely used in sugar beet crops. Therefore the investigation of genotoxic potential of Chloridazon via its interaction with DNA and evaluation of DNA damage is very important. In this study, the DNA binding properties in HEPES buffer (pH 7.2), has been monitored as a function of Chloridazon—DNA molar ratio, by spectrophotometry and spectopolarimetry. It is found that Chloridazon molecules perhaps inserts between base