Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) to 10(7) M(-1) s(-1). In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.     

Two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H nitroreductase: description of quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) M(-1) s(-1) to 10(7) M(-1) s(-1), and oxidizing 2 moles NADH per mole mononitrocompound. Oxidation of excess NADH by polynitrobenzenes including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron- or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.     

Enhancement of nitroaromatic compounds anaerobic biotransformation using a novel immobilized redox mediator prepared by electropolymerization

Functionalized polypyrrole (PPy) composites were prepared by incorporation of a model redox mediator, anthraquinonedisulphonate (AQDS), as doping anion during the electropolymerization of pyrrole (Py) monomer on active carbon felt (ACF) electrode. Then, the resulting composite, ACF/PPy/AQDS as a novel immobilized redox mediator for catalyzing anaerobic biotransformation of the model nitroaromatic compounds (NACs), such as nitrobenzene (NB), 2,4- and 2,6-dinitrotoluene (DNT), were investigated in detail. The results showed that ACF/PPy/AQDS exhibited good catalytic activity and stability, and its addition effectively accelerated the NACs anaerobic reduction to the corresponding amino compounds. In order to estimate the relationship between community dynamics and the function of immobilized redox mediator, a combined method based on fingerprints (ribosomal intergenic spacer analysis, RISA) and 16S rRNA gene sequencing was used. The results indicated that the existence of ACF/PPy/AQDS made the potent AQDS-reducing bacteria keeping predominant in the catalytic systems. Based on the results above, it can be concluded that this novel immobilized redox mediator is feasible and potentially useful to enhance NACs anaerobic reduction.     

Degradation of chlorinated nitroaromatic compounds

Chlorinated nitroaromatic compounds (CNAs) are persistent environmental pollutants that have been introduced into the environment due to the anthropogenic activities. Bacteria that utilize CNAs as the sole sources of carbon and energy have been isolated from different contaminated and non-contaminated sites. Microbial metabolism of CNAs has been studied, and several metabolic pathways for degradation of CNAs have been proposed. Detoxification and biotransformation of CNAs have also been studied in various fungi, actinomycetes and bacteria. Several physicochemical methods have been used for treatment of wastewater containing CNAs; however, these methods are not suitable for in situ bioremediation. This review describes the current scenario of the degradation of CNAs.     

Effect of polyhydric alcohols on kinetic parameters of enzymes

The effect of glycerol and other polyhydric compounds on the kinetic parameters of sixteen enzymes has been investigated. Substantial changes in K_m and turnover rate were observed. Kinetic values obtained depended on pH as well as on the specific polyhydric compound added and its concentration. No pattern in direction or extent of change in kinetic constants was evident.     

Degradation of nitroaromatic compounds by microorganisms

Nitroaromatic compounds are abundantly present in nature, but are in most cases highly toxic to living organisms. Several microorganisms, however, are capable of mineralizing or converting these compounds. Until now four pathways for the complete degradation of nitroaromatics have been described, which start with either the oxygenolytic or reductive removal of the nitro group from the aromatic ring or with this removal by means of replacement reactions. Besides these conversions many organisms are able to reduce nitroaromatics. The degradation of nitroaromatic compounds does not only occur in pure cultures but also in situ, for example in soil, water and sewage. However, several problems are associated with the application of microorganisms in the bioremediation of contaminated sites, as nitroaromatics or their conversion products may chemically interact with soil particles and cells. Besides the possibilities of applying microorganisms in the cleaning of sites contaminated with nitroaromatics, the use of microorganisms or enzymes in the biocatalytic production of industrially valuable products from nitroaromatics is also discussed.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Antiplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase

Prooxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of reactive oxygen species or to their inhibition of antioxidant enzyme glutathione reductase (GR, EC 1.6.4.2). We have examined the activity in vitro against Plasmodium falciparum of 24 prooxidant compounds of different structure (nitrobenzenes, nitrofurans, quinones, 1,1'-dibenzyl-4,4'-bipyridinium, and methylene blue), which possess a broad range of single-electron reduction potentials (E(1)(7)) and erythrocyte glutathione reductase inhibition constants (K(i(GR))). For a series of homologous derivatives of 2-(5'-nitrofurylvinyl)quinoline-4-carbonic acid, the relationship between compound K(i(GR)) and concentration causing 50% parasite growth inhibition (IC(50)) was absent. For all the compounds examined in this study, the dependence of IC(50) on their K(i(GR)) was insignificant. In contrast, IC(50) decreased with an increase in E(1)(7) and positive electrostatic charge of aromatic part of molecule (Z): log IC(50) (microM) = -(0.9846 +/- 0.3525) - (7.2850 +/- 1.2340) E(1)(7) (V) - (1.1034 +/- 0.1832) Z (r(2) = 0.8015). The redox cycling activity of nitroaromatic and quinoidal compounds in ferredoxin:NADP(+) reductase-catalyzed reaction and the rate of oxyhemoglobin oxidation in lysed erythrocytes increased with an increase in their E(1)(7) value. Our findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form reactive oxygen species, and much less significantly by the GR inhibition.     

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated.We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo^VI/V potential lowered by ca. 60–80 mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT^WT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K_Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.     

Effect of evolution on the kinetic properties of enzymes

1. The likely effect of a selective pressure in the direction of higher reaction fluxes on rate parameters for enzyme reactions confirming to Michaelis-Menten kinetics has been analyzed on the basis of relationships which take into account the changes in metabolite concentrations that must be associated with mutational changes of the kinetic properties of enzymes participating in metabolic pathways. 2. Arguments are presented to show that such a pressure should tend to increase kcat, whereas Km may decrease or increase depending on what stage of evolutionary development the enzyme has reached. While the early evolution of enzymes must have been associated with decreasing Km values, an increase of both kcat and Km is mandatory for enhancement of the rate performance of extensively developed enzymes which exhibit kcat/Km ratios approaching the diffusion-control limit. The latter limit is dependent on the equilibrium constant for the catalysed reaction. 3. Enzymes which have reached the diffusion-control limit for their second-order rate performance cannot be considered as perfectly evolved catalysts, but may well undergo further development towards a higher catalytic efficiency in response to the improvement of other enzymes in the metabolic pathway with regard to the criterion of an enhanced reaction flux. Such evolution is associated with an increase of the metabolite levels in the pathway, and a simple model system is examined in order to illustrate the ultimate limits for the metabolite levels and reaction flux that may obtain. 4. The theoretical evidence presented lends no support to previous proposals that certain enzymes (e.g. triosephosphate isomerase), or enzymes showing certain kinetic characteristics (e.g. kcat/Km quotients approaching 10(9) s-1 M-1), have reached the end of their evolutionary development. A claim that any specific enzyme has reached catalytic perfection would provide the unreasonable inference that all enzymes participating in intermediary metabolism have reached catalytic perfection.     

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.     

Structures of redox enzymes

There is an ever increasing flood of structural information and over 1,000 protein structures have been deposited in the Protein Data Base between January 1999 and January 2000. Major advances in the past year in the field of redox enzymes have included the structures of nitric oxide synthases in ligand-free and ligand-bound complexes, and the determination of the multi-subunit mitochondrial bc1 complex. The first,structures of flavocytochrome have also appeared providing insight into novel electron and proton pathways.     

Synthesis, electrochemistry, and molluscicidal activity of nitroaromatic compounds: effects of substituents and the role of redox potential

Abstract-Molluscicidal bioassays and electrochemical studies (measurement of first wave reduction potential, Epcl) were performed on several synthetic nitroaromatics, in relation to possible correlation between biological activity, redox potential and structural effects. Five of them presented a significant molluscicidal activity on Biomphalaria glabrata (LD50 < 20 ppm). The Epc1 values ranged from -0.532 to -0.857 V versus Ag/AgCl (0.1 M) (-0.260 to -0.585 V versus NHE), all of them, in the favorable range for reduction in vivo. Data comparison between Epc1 and molluscicidal activity indicates that the presence of the electroactive nitro group is important for the biological activity. Correlation with redox potential, however, was not evident. Structural effects seem to be the most important parameter. Higher activity is noticeable for phenols, including the para-nitro azo or hydrazo-containing compounds. No activity was observed for compounds having the benzylic substituent in meta position to the nitro group. These results suggest that activity undoubtedly involves more than reduction characteristics and that the possible formation of electrophilic species, after nitro reduction, can play an important role in molluscicidal activity against B. glabrata.     

Microbial transformation of nitroaromatic compounds in sewage effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

Heterogeneity of kinetic parameters of enzymes in situ in rat liver lobules

In the present review, metabolic compartmentation in liver lobules is discussed as being dynamic and more complex than thus far assumed on the basis of numbers of mRNA or protein molecules or the capacity (zero-order activity) of enzymes. Isoenzyme distribution patterns and local kinetic parameters of enzymes may vary over the different zones of liver lobules. As a consequence, metabolic fluxes in vivo at physiological substrate concentrations may be completely different from those that are assumed on the basis of the number of molecules or the capacity of enzymes present in zones of liver lobules. For a more correct estimation of the levels of metabolic processes in the different compartments of liver tissue, local kinetic parameters and substrate concentrations have to be determined to calculate local metabolic fluxes. direct measurements of metabolic fluxes in vivo with the use of noninvasive techniques is a promising alternative and the techniques will become increasingly important in future metabolic research.This paper was presented at the symposium Metabolic Zonation of the Liver: New Answers to Old Questions held in honour of Prof. Dr. D. Sasse's 60th birthday, 26 August 1994, in Basel     

Altered kinetic properties of rat heart mitochondrial enzymes following experimental-thyrotoxicosis

Effects of T_3- and T₄-induced thyrotoxicosis on temperature-dependent Arrhenius kinetics of succinate oxidase and Mg²⁺- and Mg²⁺ + 2,4-dinitrophenol-dependent ATPase activities in rat heart mitochondria were examined, For succinate oxidase system, treatment with T₃ and T₄ caused increase in the energy of activation in high temperature range in a dose-dependent manner. For low temperature range, increase in energy of activation was apparent only with higher doses of the hormones; with low doses a small but reproducible decrease was evinced. The phase transition temperature decreased significantly under these conditions. For the Mg²⁺- and Mg²⁺+2,4-dintro-phenol-dependent-ATPase activities, the activation energy values in high temperature range decreased in general. Activation energy values in low temperature range recorded a generalized increase in the Mg²⁺-ATPase enzyme system while the value did not change significantly for the Mg²⁺ + 2,4-dinitrophenol-ATPase; phase transition temperature registered a small but reproducible decrease under these conditions. The results are suggestive of increased membrane fluidization possibly through increased proportion of unsaturated fatty acids. The differential effects seen for succinate oxidase and ATPase systems are consistent with different lipid protein domains of these enzyme systems.     

Microbial transformation of nitroaromatic compounds in sewage effluent

[This corrects the article on p. 1239 in vol. 45.].     

Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes

Values ofK _m were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal1-4GlcNAc 2-6sialyltransferase and Gal1-3(4)GlcNAc 2-3sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal1-3GalNAc 2-3sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values ofK _m were determined using rat and human asialo₁ acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal1-3(4)GlcNAc 2-3sialyltransferase. Antifreeze glycorprotein was used as the macromolecular acceptor for the porcine enzyme. Values forK _m were also determined using CMP-NeuAc as the variable substrate.Abbreviations NeuAc N-acetylneuraminic acid - Gal galactose - GlcNAc N-acetylglucosamine Enzymes: Gal1-4GlcNAc 2-6sialyltransferase, EC 2.4.99.1; Gal1-3(4)GlcNAc 2-3sialyltransferase, EC 2.4.99.5; Gal1-3GalNAc 2-3sialyltransferase, EC 2.4.99.4.