Phthalate dioxygenase (PDO) and its reductase (PDR) are parts of a two-component Rieske oxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate. Aspartate D178 in PDO, which lies between the Rieske [2Fe-2S] center of one subunit and the mononuclear center of the adjacent subunit, is highly conserved among the Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer in naphthalene dioxygenase and in substrate binding and oxygen reactivity in anthranilate dioxygenase. Substitution of D178 with alanine or asparagine in PDO resulted in proteins with significantly increased Fe(II) dissociation constants. The rates of oxidation of the reduced Rieske centers in D178A and D178N were decreased by more than 10(4)-fold; only part of the loss of activity can be attributed to depletion of iron from the mononuclear centers. Reduction of PDO by reduced PDR was also slower in the D178A and D178N variants. Observed decreases in turnover rates of D178A and D178N compared to that of wild-type (WT) PDO (>10(2)-fold) can be ascribed to the cumulative effect of the low intrinsic iron content of the D178A and D178N mutants and the combination of the decreased rates of Rieske center reduction and oxidation. The coupling of dihydrodiol formation approached 100% in WT PDO but was only approximately 16% in D178A and approximately 7% in D178N. In single-turnover experiments, very small amounts of DHD were produced by D178A and D178N "as purified". The presence of saturating amounts of ferrous ion improved coupling to nearly 100% for the D178N variant but only slightly improved coupling for D178A. Thus, although hydroxylation is still possible in the variants, the reactions are largely uncoupled due to slow intramolecular electron transfer rates and the apparent weak binding of iron at the mononuclear centers.
Phthalate dioxygenase (PDO) and its reductase are parts of a two-component Rieske dioxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). Aspartate D178 in PDO, located near its ferrous mononuclear center, is highly conserved among Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer between the mononuclear iron and Rieske center in naphthalene dioxygenase (Parales, R.E. et. al. (1999) J Bacteriol 181, 1831-1837 and in substrate binding and oxygen reactivity in anthranilate dioxygenase (Beharry, Z.M. et. al (2003), Biochemistry 42, 13625-13636). The effects of substituting D178 in PDO with alanine or asparagine on the reactivity of the Rieske centers, phthalate hydroxylation, and coupling of Rieske center oxidation to DHD formation were studied previously (Pinto, A., Tarasev, M., and Ballou, D. P. (2006) Biochemistry in press). This work describes effects that D178N and D178A substitutions have on the interactions between the Rieske and mononuclear centers in PDO. The mutations affected protonation of the Rieske center histidine and conformation of subunits within the PDO multimer to create a more open structure with more solvent-accessible Rieske centers. When the Rieske centers in PDO were oxidized, D178N and D178A substitutions disrupted communication between the Rieske and Fe-mononuclear centers. This was shown by the lack of perturbations of the UV-vis spectra on phthalate binding to the D178N and D178A variants, as opposed to that observed in WT PDO. However, when the Rieske center was in the reduced state, communication between the centers was not disrupted. Phthalate binding similarly affected the rates of oxidation of the reduced Rieske center in both WT and mutant PDO. Nitric Oxide binding at the Fe(II) mononuclear center, as detected by EPR spectrometry of the Fe(II) nitrosyl complex, was regulated by the redox state of the Rieske center. When the Rieske center was oxidized in either WT or D178N PDO, NO bound to the mononuclear iron in the presence or absence of phthalate. However, when the Rieske center was reduced, NO bound only when phthalate was present. These findings are discussed in terms of the "communication functions" performed by the bridging Asp-178.Phthalate dioxygenase (PDO) and its reductase are parts of a two-component enzyme system (PDS) in Burkholderia cepacia DB01 that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). PDS comprises the monomeric phthalate dioxygenase reductase (PDR), an enzyme that contains both FMN and a plant-type [2Fe-2S] ferredoxin, and a Rieske dioxygenase (PDO), an α 6 multimer that contains Rieske and ferrous mononuclear centers. Mononuclear iron and Rieske centers on each subunit in Rieske oxygenases are shown to be separated by more than 40 Å (2), making direct electron *To whom correspondence should be addressed. Email: dballou@umich.edu NIH Public Access transfer unfavorable (3). However, as s...
Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II) -mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at ∼30 mg of crude PDO and ∼1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to Escherichia coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 hours) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was < 8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.Among the various systems used for the expression of recombinant proteins, Escherichia coli has the advantage of being available in a wide array of mutant host strains, the ability to grow rapidly and to high density, of being better characterized genetically than other microorganisms, of having many compatible expression plasmids, and it gives good yields of target proteins. Considerable attention has been paid to the improvement of E. coli systems for expression of complex eukaryotic proteins (1,2). However, expression has been especially troublesome for proteins containing Fe-S clusters that are involved in a number of physiological processes including catalysis, electron transfer, biosynthesis, DNA repair and transcriptional regulation, and sensing for regulatory processes (3). In many cases supplementation of the growth media with Fe 2+ and S 2− was not sufficient for good expression (3). The formation of Fe-S clusters was found to depend on the so-called isc (iron-sulfur cluster) genes (4,5), all nine individual components of which are important for the expression of some active ). In a number of cases it was found that coexpression of the isc cluster was essential for achieving high yields of active recombinant Fe-S proteins (11-13). Cluster inactivation in E. coli resulted in a marked decrease in the production of native and recombinant iron-sulfur * To whom correspondence should be addressed. Email: dballou@umich.edu Publisher's Dis...
L-Lysine is shown to yield an adduct with the quinone methide intermediate formed during the horseradish peroxidase (HRP)-catalyzed aerobic oxidation of eugenol (4-allyl-2-methoxyphenol). Adduct formation is evidenced by (i) lysine quenching of the characteristic quinone methide absorption band measured at 350 nm; arginine and gamma-aminobutyric acid, but not alanine or propionic acid showed similar behaviour (ii) lysine-promoted a 400 mV decrease of the eugenol oxidation voltammetric wave (1.00 V), concomitantly with an increase in current intensity and (iii) reverse phase HPLC isolation of the lysine eugenol adduct, followed by GC-MS analysis. The MS spectrum is consistent with a 2:1 lysine:eugenol adduct (MW = 455). If operative in vivo, binding of lysine to eugenol might lead to protein inactivation and possibly be involved in eugenol toxicity.
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