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Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.

An RNA-dependent RNA polymerase ribozyme that was highly optimized through in vitro evolution for the ability to copy a broad range of template sequences exhibits promiscuity toward other nucleic acids and nucleic acid analogues, including DNA, threose nucleic acid (TNA), and arabinose nucleic acid (ANA). By operating on various RNA templates, the ribozyme catalyzes multiple successive additions of DNA, TNA, or ANA monomers, although with reduced efficiency compared to RNA monomers. The ribozyme can also copy DNA or TNA templates to complementary RNAs, and to a lesser extent it can operate when both the template and product strands are composed of DNA, TNA, or ANA. These results suggest that polymerase ribozymes, which are thought to have replicated RNA genomes during the early history of life, could have transferred RNA-based genetic information to and from DNA, enabling the emergence of DNA genomes prior to the emergence of proteins. In addition, genetic systems based on nucleic acid-like molecules, which have been proposed as precursors or contemporaries of RNA-based life, could have been operated upon by a promiscuous polymerase ribozyme, thus enabling the evolutionary transition between early genetic systems.

5-Deoxyribose is formed from 5'-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by the Bacillus thuringiensis cluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite.

5-Deoxyribose is formed from 5'-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by the Bacillus thuringiensis cluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite.

Life-threatening acute liver failure can be triggered by a variety of factors, including common drugs such as acetaminophen. Positron emission tomography (PET) is rarely used to monitor liver function, in part because of a lack of specific imaging agents for liver function. Here we report a new PET probe, 2-deoxy-2-[(18)F]fluororibose ([(18)F]-2-DFR), for use in imaging liver function. [(18)F]-2-DFR was synthesized and validated as a competitive substrate for the ribose salvage pathway. [(18)F]-2-DFR was prepared through an efficient late stage radiofluorination. The desired selectivity of fluorination was achieved using an unorthodox protecting group on the precursor, which could withstand harsh SN2 reaction conditions with no side reactions. [(18)F]-2-DFR accumulated preferentially in the liver and was metabolized by the same enzymes as ribose. [(18)F]-2-DFR could distinguish between healthy liver and liver damaged by acetaminophen. [(18)F]-2-DFR is expected to be a useful PET probe for imaging and quantifying liver functions in vivo, with likely significant clinical utility.

Life-threatening acute liver failure can be triggered by a variety of factors, including common drugs such as acetaminophen. Positron emission tomography (PET) is rarely used to monitor liver function, in part because of a lack of specific imaging agents for liver function. Here we report a new PET probe, 2-deoxy-2-[(18)F]fluororibose ([(18)F]-2-DFR), for use in imaging liver function. [(18)F]-2-DFR was synthesized and validated as a competitive substrate for the ribose salvage pathway. [(18)F]-2-DFR was prepared through an efficient late stage radiofluorination. The desired selectivity of fluorination was achieved using an unorthodox protecting group on the precursor, which could withstand harsh SN2 reaction conditions with no side reactions. [(18)F]-2-DFR accumulated preferentially in the liver and was metabolized by the same enzymes as ribose. [(18)F]-2-DFR could distinguish between healthy liver and liver damaged by acetaminophen. [(18)F]-2-DFR is expected to be a useful PET probe for imaging and quantifying liver functions in vivo, with likely significant clinical utility.

Four hundred twenty-eight high-resolution DNA– protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete pi- contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either pi–pi stacking, Pi–pi T-shaped or sugar– pi contacts. Three hundred forty-four nucleobase– amino acid pi–pi contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–pi contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···pi and/or lone–pair···pi interactions, contain any amino acid and can be classified according to sugar atoms involved. Both pi–pi and sugar–pi interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein pi-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Four hundred twenty-eight high-resolution DNA– protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete pi- contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either pi–pi stacking, Pi–pi T-shaped or sugar– pi contacts. Three hundred forty-four nucleobase– amino acid pi–pi contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–pi contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···pi and/or lone–pair···pi interactions, contain any amino acid and can be classified according to sugar atoms involved. Both pi–pi and sugar–pi interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein pi-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Four hundred twenty-eight high-resolution DNA– protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete pi- contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either pi–pi stacking, Pi–pi T-shaped or sugar– pi contacts. Three hundred forty-four nucleobase– amino acid pi–pi contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–pi contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···pi and/or lone–pair···pi interactions, contain any amino acid and can be classified according to sugar atoms involved. Both pi–pi and sugar–pi interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein pi-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Four hundred twenty-eight high-resolution DNA– protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete pi- contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either pi–pi stacking, Pi–pi T-shaped or sugar– pi contacts. Three hundred forty-four nucleobase– amino acid pi–pi contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–pi contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···pi and/or lone–pair···pi interactions, contain any amino acid and can be classified according to sugar atoms involved. Both pi–pi and sugar–pi interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein pi-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Four hundred twenty-eight high-resolution DNA– protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete pi- contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either pi–pi stacking, Pi–pi T-shaped or sugar– pi contacts. Three hundred forty-four nucleobase– amino acid pi–pi contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–pi contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···pi and/or lone–pair···pi interactions, contain any amino acid and can be classified according to sugar atoms involved. Both pi–pi and sugar–pi interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein pi-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Purpose: To investigate the implications of using interaction cross sections of liquid water for the target volume when studying radiation action at the DNA level by particle track structure simulations. Materials and methods: Absolute interaction cross sections for low energy electrons between 20 eV and 1 keV were measured for tetrahydrofuran (THF), which is a substitute for deoxyribose. From these data a complete interaction cross section data set was derived and integrated in our PTB Track structure Monte Carlo code 'PTra'. Simulations of electron track structure in THF and water were performed and ionisation cluster size distributions in nanometric target volumes were determined. From these a nanodosimetric estimate for the probability to produce a double strand break was derived. Results: The probability distribution of ionisation cluster sizes was found to be shifted towards smaller values for a THF-filled target as compared to a water-filled one. For all electron energies investigated, the nanodosimetric estimates for double-strand break probability in the THF-filled target have lower values than for a target of liquid water. Conclusion: The preliminary results indicate that simulations based on cross sections of water would overestimate the initial direct radiation damage to the DNA.

Purpose: To investigate the implications of using interaction cross sections of liquid water for the target volume when studying radiation action at the DNA level by particle track structure simulations. Materials and methods: Absolute interaction cross sections for low energy electrons between 20 eV and 1 keV were measured for tetrahydrofuran (THF), which is a substitute for deoxyribose. From these data a complete interaction cross section data set was derived and integrated in our PTB Track structure Monte Carlo code 'PTra'. Simulations of electron track structure in THF and water were performed and ionisation cluster size distributions in nanometric target volumes were determined. From these a nanodosimetric estimate for the probability to produce a double strand break was derived. Results: The probability distribution of ionisation cluster sizes was found to be shifted towards smaller values for a THF-filled target as compared to a water-filled one. For all electron energies investigated, the nanodosimetric estimates for double-strand break probability in the THF-filled target have lower values than for a target of liquid water. Conclusion: The preliminary results indicate that simulations based on cross sections of water would overestimate the initial direct radiation damage to the DNA.

Purpose: To investigate the implications of using interaction cross sections of liquid water for the target volume when studying radiation action at the DNA level by particle track structure simulations. Materials and methods: Absolute interaction cross sections for low energy electrons between 20 eV and 1 keV were measured for tetrahydrofuran (THF), which is a substitute for deoxyribose. From these data a complete interaction cross section data set was derived and integrated in our PTB Track structure Monte Carlo code 'PTra'. Simulations of electron track structure in THF and water were performed and ionisation cluster size distributions in nanometric target volumes were determined. From these a nanodosimetric estimate for the probability to produce a double strand break was derived. Results: The probability distribution of ionisation cluster sizes was found to be shifted towards smaller values for a THF-filled target as compared to a water-filled one. For all electron energies investigated, the nanodosimetric estimates for double-strand break probability in the THF-filled target have lower values than for a target of liquid water. Conclusion: The preliminary results indicate that simulations based on cross sections of water would overestimate the initial direct radiation damage to the DNA.

Purpose: To investigate the implications of using interaction cross sections of liquid water for the target volume when studying radiation action at the DNA level by particle track structure simulations. Materials and methods: Absolute interaction cross sections for low energy electrons between 20 eV and 1 keV were measured for tetrahydrofuran (THF), which is a substitute for deoxyribose. From these data a complete interaction cross section data set was derived and integrated in our PTB Track structure Monte Carlo code 'PTra'. Simulations of electron track structure in THF and water were performed and ionisation cluster size distributions in nanometric target volumes were determined. From these a nanodosimetric estimate for the probability to produce a double strand break was derived. Results: The probability distribution of ionisation cluster sizes was found to be shifted towards smaller values for a THF-filled target as compared to a water-filled one. For all electron energies investigated, the nanodosimetric estimates for double-strand break probability in the THF-filled target have lower values than for a target of liquid water. Conclusion: The preliminary results indicate that simulations based on cross sections of water would overestimate the initial direct radiation damage to the DNA.

Cyclohexene nucleic acids (CeNA), which are characterized by the presence of a cyclohexene moiety instead of a natural (deoxy)ribose sugar, are known to increase the thermal and enzymatic stability when incorporated in RNA oligonucleotides. As it has been demonstrated that even a single cyclohexenyl nucleoside, when incorporated in an oligonucleotide, can have a profound effect on the biological activity of the oligonucleotide, further research is warranted to study the complex of such oligonucleotides with target proteins. In order to analyse the influence of CeNA residues onto the helix conformation and hydration of natural nucleic acid structures, a cyclohexenyl-adenine building block (xAr) was incorporated into the Dickerson sequence CGCGA(xAr)TTCGCG. The crystal structure of this sequence determined to a resolution of 1.90 Å. The global helix belongs to the B-type family and shows a water spine, which is partially broken up by the apolar cyclohexene residue. The cyclohexene ring adopts the 2 E-conformation allowing a better incorporation of the residue in the dodecamer sequence. The crystal packing is stabilized by cobalt hexamine residues and belongs to space group P2221, never before reported for nucleic acids. © 2007 The Author(s).

CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 Å, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3′-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking. © 2008 American Chemical Society.

The lung toxicity of a carbide-cobalt mixture is more important than that of each individual component; the mechanism of this interaction is not understood. The capacity of cobalt metal particles alone and mixed with different carbides to generate hydroxyl radicals was examined with the deoxyribose assay. In a chemical system, cobalt ions and cobalt metal particles (Co) were found to catalyse the degradation of deoxyribose in the presence of hydrogen peroxide. Carbides were able to directly oxidize deoxyribose, but their respective activities did not support such a mechanism to explain the carbide-cobalt interactive toxicity, since there was no direct relationship between deoxyribose degradation ability and cytotoxicity toward macrophages. Tungsten, niobium, titanium and chromium carbides (interactive carbides) were only weak oxidants and conversely molybdenum, vanadium and silicon carbides (non-interactive carbides) were the most potent ones. The ability of cobalt metal to produce hydroxyl radicals in the presence of hydrogen peroxide was not increased by tungsten carbide. The role of reactive radical formation in the toxicity of these particles was further assessed in a macrophage culture model. Catalase (4000 U/ml), superoxide dismutase (300 U/ml), sodium azide (1 mM), sodium benzoate, mannitol, taurine and methionine (all 20 mM) were all unable to protect against the cytotoxic effects of cobalt ions and cobalt metal alone or mixed with tungsten carbide. In conclusion, no evidence was found that production of reactive oxygen species contributes to the elective toxicity of carbide-cobalt mixtures.