Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

Nuclear DNA topoisomerase I (TOP1) is an essential human enzyme. It is the only known target of the alkaloid camptothecin, from which the potent anticancer agents irinotecan and topotecan are derived. As camptothecins bind at the interface of the TOP1-DNA complex, they represent a paradigm for interfacial inhibitors that reversibly trap macromolecular complexes. Several camptothecin and non-camptothecin derivatives are being developed to further increase anti-tumour activity and reduce side effects. The mechanisms and molecular determinants of tumour response to TOP1 inhibitors are reviewed, and rational combinations of TOP1 inhibitors with other drugs are considered based on current knowledge of repair and checkpoint pathways that are associated with TOP1-mediated DNA damage.

Topoisomerase I inhibitors : camptothecins and beyond

Antibiotic drug-target interactions, and their respective direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochemical pathways. In this Review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, could be exploited to create new antibacterial therapies.

How antibiotics kill bacteria: from targets to networks

Autism is a heterogeneous syndrome defined by impairments in three core domains: social interaction, language and range of interests. Recent work has led to the identification of several autism susceptibility genes and an increased appreciation of the contribution of de novo and inherited copy number variation. Promising strategies are also being applied to identify common genetic risk variants. Systems biology approaches, including array-based expression profiling, are poised to provide additional insights into this group of disorders, in which heterogeneity, both genetic and phenotypic, is emerging as a dominant theme.

/pdf/advances-in-autism-genetics-on-the-threshold-of-a-new-1vbpm6dbmd.pdf

Advances in autism genetics: on the threshold of a new neurobiology

Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.

Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.

https://www.pnas.org/content/pnas/73/11/3872.full.pdf

DNA gyrase: an enzyme that introduces superhelical turns into DNA

Crystal structure of an ihf-dna complex : a protein-induced dna u-turn

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

https://www.pnas.org/content/pnas/93/21/11534.full.pdf

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

E. coli integration host factor binds to specific sites in DNA

Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.

/pdf/yeast-gene-for-a-tyr-dna-phosphodiesterase-that-repairs-3nyhdpfsec.pdf

Howard A. Nash

Papers

DNA gyrase: an enzyme that introduces superhelical turns into DNA

Crystal structure of an ihf-dna complex : a protein-induced dna u-turn

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

E. coli integration host factor binds to specific sites in DNA

Yeast Gene for a Tyr-DNA Phosphodiesterase that Repairs Topoisomerase I Complexes