We report the design, construction, and testing of Escherichia coli-based bioluminescent biorepor... more We report the design, construction, and testing of Escherichia coli-based bioluminescent bioreporters for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX), one of the most prevalent military-grade explosives in use today. These sensor strains are based on a fusion between the promoter of either the hmp (nitric oxide dioxygenase) or the hcp (a high-affinity nitric oxide reductase) E. coli gene, to the microbial bioluminescence luxCDABEG gene cassette. Signal intensity was enhanced in ∆hmp and ∆hcp mutants, and detection sensitivity was improved when the two gene promoters were cloned in tandem. The Photobacterium leiognathi luxCDABEG reporter genes were superior to those of Aliivibrio fischeri in terms of signal intensity, but in most cases inferior in terms of detection sensitivity, due to a higher background signal. Both sensor strains were also induced by additional nitro-organic explosives, as well as by nitrate salts. Sensitive detection of RDX in a solid matrix (either LB agar or sand) was also demonstrated, with the bioreporters encapsulated in 1.5-mm calcium alginate beads. Lowest RDX concentration detected in sand was 1.67 mg/kg sand. The bioreporter strains described herein may serve as a basis for a standoff detection technology of RDX-based explosive devices, including buried landmines.
DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based... more DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in E. coli , have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells. IMPORTANCE The information provided in this article sheds new light both on the microbi...
Mitochondria are the main source of ATP production and also contribute to many other processes ce... more Mitochondria are the main source of ATP production and also contribute to many other processes central to cellular function. Mitochondrial activities have been linked with growth, differentiation and aging. As relicts of bacterial endosymbionts, these organelles contain their own genetic system (i.e., mitogenome or mtDNA). The expression of the mtDNA in plants is complex, particularly at the posttranscriptional level. Following transcription, the polycistronic pre-RNAs in plant mitochondria are processed into individual RNAs, which then undergo extensive modifications, as trimming, splicing and C-to-U editing, before being translated by organellar ribosomes. Our study focuses on N6-methylation of Adenosine ribonucleotides (m6A-RNA) in plant mitochondria. m6A is the most common modification in eukaryotic mRNAs. The biological significance of this highly dynamic modification is under investigation, but it is widely accepted that m6A mediates structural switches that affect RNA stabili...
Applied and environmental microbiology, Jan 8, 2017
DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potent... more DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT, and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study we have characterized the initial stages of DNT biotransformation in E. coli, have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite which induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source, and propose that this compound is metabolized in order to neutralize its toxicity to the cells.Importance The information provided in this article sheds new light both on the microbial biodegradability of n...
Elevated levels of activated protein kinase B (PKB/Akt) have been detected in many types of cance... more Elevated levels of activated protein kinase B (PKB/Akt) have been detected in many types of cancer. Substrate-based peptide inhibitors have the advantage of selectivity due to their extensive interactions with the kinase-specific substrate binding site but often lack necessary pharmacological properties. Chemical modifications of potent peptide inhibitors, such as cyclization, may overcome these drawbacks while maintaining potency. We present an extensive structure-activity relationship (SAR) study of a potent peptide-based PKB/Akt inhibitor. Two backbone cyclic (BC) peptide libraries with varying modes of cyclization, bridge chemistry, and ring size were synthesized and evaluated for in vitro PKB/Akt inhibition. Backbone-to-backbone urea BC peptides were more potent than N-terminus-to-backbone amide BC peptides. Several analogues were up to 10-fold more active than the parent linear peptide. Some activity trends could be rationalized using computational surface mapping of the PKB/Akt kinase catalytic domain. The novel molecules have enhanced pharmacological properties which make them promising lead candidates.
Although Cpd I appears to be elusive and has never been detected experimentally, there is indirec... more Although Cpd I appears to be elusive and has never been detected experimentally, there is indirect evidence that confirm its existence.6 These studies have led to the suggestion that the hydroxylation of aliphatic substrates by Cpd I proceeds via the hydrogen abstraction/oxygen rebound ...
DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and... more DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and propene by Compound I of P450cam demonstrate that the relative barriers for the oxidative processes themselves are not good predictors of the observed selectivity. However, a kinetic expression previously developed (Kozuch, S.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 3355) for catalytic cycles under steady-state conditions, predicts, in accord with experiment, that propene will undergo exclusive C=C epoxidation, while cyclohexene will undergo both reactions with a small preference for epoxidation. The model expression for the effective barrier of the cycle forms a general basis for understanding and predicting the selectivity of P450 isozymes.
Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally ... more Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C-H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme.
We report the design, construction, and testing of Escherichia coli-based bioluminescent biorepor... more We report the design, construction, and testing of Escherichia coli-based bioluminescent bioreporters for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX), one of the most prevalent military-grade explosives in use today. These sensor strains are based on a fusion between the promoter of either the hmp (nitric oxide dioxygenase) or the hcp (a high-affinity nitric oxide reductase) E. coli gene, to the microbial bioluminescence luxCDABEG gene cassette. Signal intensity was enhanced in ∆hmp and ∆hcp mutants, and detection sensitivity was improved when the two gene promoters were cloned in tandem. The Photobacterium leiognathi luxCDABEG reporter genes were superior to those of Aliivibrio fischeri in terms of signal intensity, but in most cases inferior in terms of detection sensitivity, due to a higher background signal. Both sensor strains were also induced by additional nitro-organic explosives, as well as by nitrate salts. Sensitive detection of RDX in a solid matrix (either LB agar or sand) was also demonstrated, with the bioreporters encapsulated in 1.5-mm calcium alginate beads. Lowest RDX concentration detected in sand was 1.67 mg/kg sand. The bioreporter strains described herein may serve as a basis for a standoff detection technology of RDX-based explosive devices, including buried landmines.
DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based... more DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in E. coli , have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells. IMPORTANCE The information provided in this article sheds new light both on the microbi...
Mitochondria are the main source of ATP production and also contribute to many other processes ce... more Mitochondria are the main source of ATP production and also contribute to many other processes central to cellular function. Mitochondrial activities have been linked with growth, differentiation and aging. As relicts of bacterial endosymbionts, these organelles contain their own genetic system (i.e., mitogenome or mtDNA). The expression of the mtDNA in plants is complex, particularly at the posttranscriptional level. Following transcription, the polycistronic pre-RNAs in plant mitochondria are processed into individual RNAs, which then undergo extensive modifications, as trimming, splicing and C-to-U editing, before being translated by organellar ribosomes. Our study focuses on N6-methylation of Adenosine ribonucleotides (m6A-RNA) in plant mitochondria. m6A is the most common modification in eukaryotic mRNAs. The biological significance of this highly dynamic modification is under investigation, but it is widely accepted that m6A mediates structural switches that affect RNA stabili...
Applied and environmental microbiology, Jan 8, 2017
DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potent... more DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT, and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study we have characterized the initial stages of DNT biotransformation in E. coli, have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite which induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source, and propose that this compound is metabolized in order to neutralize its toxicity to the cells.Importance The information provided in this article sheds new light both on the microbial biodegradability of n...
Elevated levels of activated protein kinase B (PKB/Akt) have been detected in many types of cance... more Elevated levels of activated protein kinase B (PKB/Akt) have been detected in many types of cancer. Substrate-based peptide inhibitors have the advantage of selectivity due to their extensive interactions with the kinase-specific substrate binding site but often lack necessary pharmacological properties. Chemical modifications of potent peptide inhibitors, such as cyclization, may overcome these drawbacks while maintaining potency. We present an extensive structure-activity relationship (SAR) study of a potent peptide-based PKB/Akt inhibitor. Two backbone cyclic (BC) peptide libraries with varying modes of cyclization, bridge chemistry, and ring size were synthesized and evaluated for in vitro PKB/Akt inhibition. Backbone-to-backbone urea BC peptides were more potent than N-terminus-to-backbone amide BC peptides. Several analogues were up to 10-fold more active than the parent linear peptide. Some activity trends could be rationalized using computational surface mapping of the PKB/Akt kinase catalytic domain. The novel molecules have enhanced pharmacological properties which make them promising lead candidates.
Although Cpd I appears to be elusive and has never been detected experimentally, there is indirec... more Although Cpd I appears to be elusive and has never been detected experimentally, there is indirect evidence that confirm its existence.6 These studies have led to the suggestion that the hydroxylation of aliphatic substrates by Cpd I proceeds via the hydrogen abstraction/oxygen rebound ...
DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and... more DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and propene by Compound I of P450cam demonstrate that the relative barriers for the oxidative processes themselves are not good predictors of the observed selectivity. However, a kinetic expression previously developed (Kozuch, S.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 3355) for catalytic cycles under steady-state conditions, predicts, in accord with experiment, that propene will undergo exclusive C=C epoxidation, while cyclohexene will undergo both reactions with a small preference for epoxidation. The model expression for the effective barrier of the cycle forms a general basis for understanding and predicting the selectivity of P450 isozymes.
Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally ... more Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C-H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme.
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Papers by Carina Hazan