Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.
Oxidative stress is generally considered as the consequence of an imbalance between pro- and antioxidants species, which often results into indiscriminate and global damage at the organismal level. Elderly people are more susceptible to oxidative stress and this depends, almost in part, from a decreased performance of their endogenous antioxidant system. As many studies reported an inverse correlation between systemic levels of antioxidants and several diseases, primarily cardiovascular diseases, but also diabetes and neurological disorders, antioxidant supplementation has been foreseen as an effective preventive and therapeutic intervention for aging-associated pathologies. However, the expectations of this therapeutic approach have often been partially disappointed by clinical trials. The interplay of both endogenous and exogenous antioxidants with the systemic redox system is very complex and represents an issue that is still under debate. In this review a selection of recent clinical studies concerning antioxidants supplementation and the evaluation of their influence in aging-related diseases is analyzed. The controversial outcomes of antioxidants supplementation therapies, which might partially depend from an underestimation of the patient specific metabolic demand and genetic background, are presented.
This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined.The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA) 2 , endowed with hydroxylase activity.Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe)2S] cluster, and exhibits a typical flavodoxin absorption spectrum.Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected.C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe)2S] cluster. The cluster is very sensitive to oxygen damage.Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified.Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.Keywords: monooxygenase; protein expression; electron transfer; bioremediation; recombinant.Several strains from Pseudomonas genus grow on aromatic compounds due to enzymatic systems able to activate aromatic rings by mono-and di-hydroxylations and to operate ortho or meta-cleavage pathway [1,2] which leads to citric acid cycle intermediates.Toluene/o-xylene-monooxygenase (Tomo) from Pseudomonas stutzeri OX1 [3,4] is endowed with a broad spectrum of substrate specificity [3], and the ability to hydroxylate more than a single position of the aromatic ring in two consecutive monooxygenation reactions [3]. Thus Tomo is able to oxidize o-, m-and p
Cationic antimicrobial peptides (AMPs) possess fast and broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein-derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133-150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad-spectrum antibacterial activity. As shown for several other AMPs, ApoE (133-150) is structured in the presence of TFE and of membrane-mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo-polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133-150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP-1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti-inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.
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