Glutarate L-2-hydroxylase (CsiD/GlaH) is an archetype Fe(II)/2-oxoglutarate-dependent dioxygenase.

The Escherichia coli gene initially named ygaT is located adjacent to lhgO, encoding L-2-hydroxyglutarate oxidase/dehydrogenase, and the gabDTP gene cluster, utilized for γ-aminobutyric acid (GABA) metabolism. Because this gene is transcribed specifically during periods of carbon starvation, it was renamed csiD for carbon starvation induced. The CsiD protein was structurally characterized and shown to possess a double-stranded ß-helix fold, characteristic of a large family of non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases. Consistent with a role in producing the substrate for LhgO, CsiD was shown to be a glutarate L-2-hydroxylase. We review the kinetic and structural properties of glutarate L-2-hydroxylase from E. coli and other species, and we propose a catalytic mechanism for this archetype 2OG-dependent hydroxylase. Glutarate can be derived from l-lysine within the cell, with the gabDT genes exhibiting expanded reactivities beyond those known for GABA metabolism. The complete CsiD-containing pathway provides a means for the cell to obtain energy from the metabolism of l-lysine during periods of carbon starvation. To reflect the role of this protein in the cell, a renaming of csiD to glaH has been proposed.

Reduction of urease activity by interaction with the flap covering the active site.

With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors.

Biosynthesis of the urease metallocenter.

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview.

Fructose-1,6-bisphosphate aldolase (class II) is the primary site of nickel toxicity in Escherichia coli.

Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 µM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 µM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 µM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 µM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site.

Mechanisms of nickel toxicity in microorganisms.

Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: (1) nickel replaces the essential metal of metalloproteins, (2) nickel binds to catalytic residues of non-metalloenzymes; (3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically and (4) nickel indirectly causes oxidative stress.

The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity.

Excess copper is poisonous to all forms of life, and copper overloading is responsible for several human pathologic processes. The primary mechanisms of toxicity are unknown. In this study, mutants of Escherichia coli that lack copper homeostatic systems (copA cueO cus) were used to identify intracellular targets and to test the hypothesis that toxicity involves the action of reactive oxygen species. Low micromolar levels of copper were sufficient to inhibit the growth of both WT and mutant strains. The addition of branched-chain amino acids restored growth, indicating that copper blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme in this pathway. Other enzymes in this iron-sulfur dehydratase family were similarly affected. Inactivation did not require oxygen, in vivo or with purified enzyme. Damage occurred concomitant with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. Copper efflux by dedicated export systems, chelation by glutathione, and cluster repair by assembly systems all enhance the resistance of cells to this metal.

Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli.

Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.

Mitochondrial iron detoxification is a primary function of frataxin that limits oxidative damage and preserves cell longevity.

Friedreich ataxia is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin. Although frataxin is ubiquitously expressed, frataxin deficiency leads to a selective loss of dorsal root ganglia neurons, cardiomyocytes and pancreatic beta cells. How frataxin normally promotes survival of these particular cells is the subject of intense debate. The predominant view is that frataxin sustains mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron-sulfur cluster (ISC) assembly and repair. We have proposed that frataxin not only promotes the biogenesis of iron-containing enzymes, but also detoxifies surplus iron thereby affording a critical anti-oxidant mechanism. These two functions have been difficult to tease apart, however, and the physiologic role of iron detoxification by frataxin has not yet been demonstrated in vivo. Here, we describe mutations that specifically impair the ferroxidation or mineralization activity of yeast frataxin, which are necessary for iron detoxification but do not affect the iron chaperone function of the protein. These mutations increase the sensitivity of yeast cells to oxidative stress, shortening chronological life span and precluding survival in the absence of the anti-oxidant enzyme superoxide dismutase. Thus, the role of frataxin is not limited to promoting ISC assembly or heme synthesis. Iron detoxification is another function of frataxin relevant to anti-oxidant defense and cell longevity that could play a critical role in the metabolically demanding environment of non-dividing neuronal, cardiac and pancreatic beta cells.

RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation.

RecA- and RecBC-catalyzed repair in eubacteria assembles chromosomes fragmented by double-strand breaks. We propose that recA mutants, being unable to repair fragmented chromosomes, depend on various strategies designed to avoid chromosomal fragmentation. To identify chromosomal fragmentation-avoidance strategies, we screened for Escherichia coli mutants synthetically inhibited in combination with recA inactivation by identifying clones unable to lose a plasmid carrying the recA(+) gene. Using this screen, we have isolated several RecA-dependent mutants and assigned them to three distinct areas of metabolism. The tdk and rdgB mutants affect synthesis of DNA precursors. The fur, ubiE, and ubiH mutants are likely to have increased levels of reactive oxygen species. The seqA, topA mutants and an insertion in smtA perturbing the downstream mukFEB genes affect nucleoid administration. All isolated mutants show varying degree of SOS induction, indicating elevated levels of chromosomal lesions. As predicted, mutants in rdgB, seqA, smtA, topA, and fur show increased levels of chromosomal fragmentation in recBC mutant conditions. Future characterization of these RecA-dependent mutants will define mechanisms of chromosomal fragmentation avoidance.

Taxis response of various denitrifying bacteria to nitrate and nitrite.

The taxis response of Rhodobacter sphaeroides 2.4.1 and 2.4.3, Rhodopseudomonas palustris, and Agrobacterium tumefaciens to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions. R. sphaeroides 2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite. R. sphaeroides 2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in R. palustris and A. tumefaciens. R. palustris exhibited a taxis response to nitrite but not to nitrate, while A. tumefaciens exhibited a response to both compounds.