The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regioand enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 Å or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the ␣ 2  2 ␥ 2 tertiary structure are notably different. Of particular interest is a 6 -10 Å-wide channel of ϳ35 Å in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.Bacterial multicomponent monooxygenases (BMMs) 1 comprise a family of carboxylate-bridged non-heme diiron enzymes capable of oxidizing a broad range of hydrocarbons including C 1 -C 8 alkanes, alkenes, and aromatics (1, 2). Four characterized subclasses of multicomponent monooxygenases have been defined (2, 3). These are soluble methane monooxygenases (sMMOs), four-component alkene/arene monooxygenases or toluene monooxygenases (TMOs), three-component phenol hydroxylases (PHs), and ␣ alkene monoxygenases (AMOs), of which all are believed to have evolved from a common ancestor. Bacteria containing multicomponent monooxygenases are capable of using specific hydrocarbon substrates as their primary source of carbon and energy (1, 2, 4). The remarkable range of substrate specificity exhibited by these enzymes endows these bacteria with the ability to bioremediate environmentally harmful substances such as trichloroethylene and petroleum spills (5, 6) and to regulate the global carbon cycle (4). BMMs can also perform regio-and stereospecific hydroxylations, making them useful for producing pure feedstocks for industrial synthesis (7). These enzyme systems, although highly homologous, have evolved different substrate specificities. Only soluble methane monooxygenase can activate the inert C-H bond of methane, which is one of the most difficult reactions to perform in nature (1), whereas the catalytic abilities of TMOs are limited to aromatics, alkenes, and some haloalkanes (2, 5).Substrate hydroxylation in BMMs occurs at a dioxygen-activated, carboxylate-bridged diiron center in the ␣-subunit of a ϳ220 -250 kDa hydroxylase component that is an (␣␥) 2 heterodimer or, in the case of one known AMO, an ␣ monomer (1-3, 8, 9). Sequence identity comparisons and spectroscopic studies suggest that the diiron centers of...
We report the results of a comparative analysis of the sequences of multicomponent monooxygenases, a family of enzymes of great interest for bioremediation of contaminated soil. We show that their function, in terms of substrate specificity, can be deduced from their subunit organization and composition, that rearrangements of subunits as well as recruitments of new ones can be used to explain their different properties and functionalities, and that the observed pattern can be rationalized invoking a number of evolutionary events, including horizontal gene transfer. Our analysis highlights the plasticity and modularity of this family of enzymes, which might very well be the reason underlying the extremely rapid emergence of new bacterial strains able to grow on contaminated soils.
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.
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
Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.
It has been previously shown that the antitumor action of bovine seminal rihonuclease (BS-RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N-terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS-RNase is the exchange of N-terminal ends between subunits.
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