A Survey of Oxidative Paracatalytic Reactions Catalyzed by Enzymes That Generate Carbanionic Intermediates: Implications for ROS Production, Cancer Etiology, and Neurodegenerative Diseases

Enzymes that generate carbanionic intermediates often catalyze paracatalytic reactions with O2 and other electrophiles not considered “normal” reactants. For example, pyridoxal 5′-phosphate (PLP)—containing pig kidney dopa decarboxylase oxidizes dopamine with molecular O2 to 3,4-dihydroxyphenylacetaldehyde at about 1% of the rate at which it catalyzes nonoxidative dopa decarboxylation. The mutant Y332F enzyme, however, catalyzes stoichiometric conversion of dopa to 3,4-dihydroxyphenylacetaldehyde, suggesting that even minor structural changes may alter or initiate paracatalytic reactions catalyzed by certain enzymes. Carbanions generated by several thiamine diphosphate (ThDP)—dependent enzymes react with different electrophiles, transforming some xenobiotics and endogenous compounds into potentially biologically hazardous products. The detrimental effects of paracatalytic reactions may be greatly increased by cellular compartmentation of enzymes and intermediates. For example, in two of the the three multienzyme complexes involved in oxidative α-keto acid decarboxylation, paracatalytic reactions of the third component inactivate the first carbanion-generating component. In this review we provide an outline of carbanion-generating enzymes known to catalyze paracatalytic reactions. We also discuss the potential of some of these reactions to contribute to irreversible damage in cancer and neurodegeneration through disease-induced alterations in the metabolic state and/or protein structure

Characterization of acetolactate synthase resistance in common sunflower (Helianthus annuus)

Herbicides that inhibit acetolactate synthase (ALS) have been important weed management tools for nearly 20 years. In recent years, resistance to ALS inhibiting herbicides has increased. In 1996, a population of common sunflower near Howard, South Dakota was suspected to be resistant to chlorimuron and imazethapyr, both ALS-inhibiting herbicides. Whole-plant ALS enzyme assays and herbicide dose response experiments concluded that cross resistance between chlorimuron and imazethapyr was present in the Howard common sunflower population. The percentages of resistant to sensitive individuals within the resistant population also indicated that the resistant population was not homogeneously resistant to either herbicide. Herbicide penetration and translocation experiments showed that resistant plants absorbed approximately 44% and 36% less 14C-chlorimuron and 14C-imazethapyr, respectively, compared to sensitive plants. Translocation of 14C did not vary. An alanine to valine substitution at amino acid position 204 of the ALS gene was found in six of seven clones from resistant plants. A frame shift occurred in Region B of the ALS gene, suggesting that multiple copies exist in the genome. Gene flow experiments suggested that resistance is due to a semi-dominant, nuclear-encoded ALS gene

Energiekonservierung über Organohalid-Respiration in Sulfurospirillum multivorans

The epsilonproteobacterium Sulfurospirillum multivorans is able to couple the reductive dechlorination of tetrachloroethene (PCE) to energy conservation via electron transport phosphorylation (organohalide respiration). The key enzyme of this anaerobic respiration is the reductive PCE dehalogenase (PceA). In this thesis, we investigated which proteins and possible other components (for example, quinones) are involved in the electron transfer between the enzymes, responsible for the oxidation of the electron donors, and PceA. In order to elucidate the PCE respiratory chain the genome of S. multivorans was sequenced and a differential proteome analysis was conducted. Involvement of quinones and a reverse electron flow were tested by inhibition studies and the quinones were identified. The experimental approaches allowed for the identification of proteins specifically produced in PCE-grown cells. Among other proteins, a quinol dehydrogenase (similar to NapGH found in nitrate respiration) was identified, which is presumably involved in electron transfer to PceA. Subsequently, the periplasmic, iron-sulfur cluster-containing component of the quinol dehydrogenase was heterologously expressed in Escherichia coli and purified. With quinone analogues, PCE respiration was inhibited, pointing to the involvement of quinones. In S. multivorans, organohalide respiration was blocked by protonophores while there was no effect of uncouplers on PCE-respiration of the gram-positive Desulfitobacterium hafniense Y51. The latter organism lacks NapGH-type quinol dehydrogenase genes. Until now, the microbial dechlorination of PCE has only be described under anaerobic conditions. In this study, the ability of the microaerobic organism S. multivorans to dechlorinate PCE in the presence of oxygen concentrations below 0.5% was demonstrated. The results of this study include key parameters for studies on reductive dehalogenation in oxic-anoxic zones of PCE-contaminated sites