Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies.

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis.

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.

Mössbauer study of 4-hydroxybutyryl-CoA dehydratase--probing the role of an iron-sulfur cluster in an overall non-redox reaction.

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyzes the dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. Although dehydration is an overall non-redox reaction, the enzyme contains FAD and Fe-S clusters. Previous work has shown that the Fe-S clusters are difficult to reduce and therefore unlikely to be redox-active in catalysis. Here, Mössbauer spectroscopy has been used to characterise the Fe-S clusters in active as well as in air-inactivated enzyme. In zero magnetic field at 80 K and 4.2 K, the spectra of active dehydratase consisted mainly of one species (95%) with quadrupole splitting, deltaE(Q) = 1.00 mm s(-1) and isomer shift, delta = 0.43 mm s(-1). Magnetically perturbed Mössbauer spectra indicated a spin of zero. In the presence of 6 mM crotonyl-CoA, the spectra remained unchanged. Taken together, the data show that there are [4Fe-4S]2+ in the enzyme, most probably two clusters/homotetramer, that the four iron atoms in each cluster are coordinated in an identical fashion, and that there is no direct interaction with substrates. We therefore infer that the Fe-S clusters serve a structural rather than a catalytic role in 4-hydroxybutyryl-CoA dehydratase. In air-inactivated enzyme (10% residual activity), a new doublet appeared (58%) with deltaE(Q) = 0.72 mm s(-1), delta = 0.32 mm s(-1) and S = 0. The assignment of this subspectrum to [3Fe-4S]+ clusters, based on the typical Mössbauer parameters, is contradicted by the finding of spin zero for the species. One possible explanation could be spin-coupling of two [3Fe-4S]+ clusters in close proximity.

Hyperthyroidism with or without pyramidal lobe Graves' disease or disseminated autonomously functioning thyroid tissue?

I-123 thyroid scintigrams performed in 349 patients were evaluated with a focus on specific thyroid gland vestiges, namely a pyramidal lobe or a thyroglossal duct. The detection of these vestiges in patients with hyperthyroidiam is indicative of autoimmune hyperthyroidism. In Graves' disease, stimulating thyrotropin (TSH) receptor antibodies cause a significantly more frequent appearance of vestiges of the thyroglossal tract. In contrast, disseminated autonomously functioning thyroid nodules rarely show a pyramidal lobe. The frequency of pyramidal lobe visualization in patients with Graves' disease differed significantly from the frequency in patients with multifocal or disseminated autonomously functioning nodules. In euthyroidism patients, the vestiges may be indicative of the diagnosis of iodine deficiency with or without latent primary hypothyroidism. In thyroid scintigraphy, the pyramidal lobe and the thyroglossal duct can be visualized more easily using I-123 instead of Tc-99m sodium pertechnetate.

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction.

4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm. The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/- 15 mV; pH 7.0, 25 degrees C). There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce. After extensive photoreduction, an EPR signal indicative of a [4Fe-4S]+ cluster was detected (g-values: 2.037, 1.895, 1.844). Upon exposure to air at 0 degrees C, the enzyme lost dehydration activity completely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day. The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond. The putative [4Fe-4S]2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group.

Lactate monooxygenase. I. Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266.

Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water. The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Brändén, C. I. (1989) J. Biol. Chem. 264, 3624-3628; Xia, Z. X., and Mathews, S. F. (1990) J. Mol. Biol. 212, 837-863). There is strong homology, especially around residues in the active site. The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN. Based on the homology, lysine 266 is the residue suggested to neutralize that charge. Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E. coli and purification procedures developed. The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M. smegmatis. Mutation of lysine 266 to a methionine created K266M. The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable. This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0. In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position. In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1). The rate of reduction with L-lactate is significantly decreased in K266M. Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme. In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme. This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge.

Lactate monooxygenase. II. Site-directed mutagenesis of the postulated active site base histidine 290.

Lactate monooxygenase catalyzes the oxidation of L-lactate with molecular oxygen to acetate, CO2, and water. Histidine 290 has been proposed to be the active site base in lactate monooxygenase (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and was mutated to a glutamine (H290Q). The mutant enzyme shows properties that support strongly the postulated function of the histidine. The ability of L-lactate to reduce the enzyme flavin is essentially abolished, whereas reoxidation of reduced enzyme with oxygen proceeds at 1.4 x 10(4) M-1 s-1, a rate essentially like that found in the wild type enzyme. The substrate, L-lactate, is bound with a Kd equal to 2.0 x 10(-2) M, and D-lactate, a competitive inhibitor with a Kd of 3.1 x 10(-3) M. Both values are similar to binding measured in the wild type enzyme. Unlike the situation with wild type enzyme, where the transition state analog oxalate is bound tightly in a two-step reaction involving proton uptake from solution (Ghisla, S., and Massey, V. (1977) J. Biol. Chem. 252, 6729-6735), the mutant enzyme binds oxalate weakly, in a single step reaction, with a Kd in the order of 0.1 M. No effect was observed upon varying the pH, indicating that binding does not include a protonation step. Replacing the histidine also has a significant effect on the ability of the enzyme to stabilize the flavin N(5)-sulfite adduct. Sulfite is bound at least 1000-fold weaker than it is in the wild type enzyme.

Lactate monooxygenase. III. Additive contributions of active site residues to catalytic efficiency and stabilization of an anionic transition state.

Lactate monooxygenase catalyzes the conversion of L-lactate to acetate, CO2, and water with incorporation of molecular oxygen. Several amino acid residues of lactate monooxygenase had been postulated to interact in specific ways with the bound substrate (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632). Tyrosine 44 and arginine 293 were proposed to form a hydrogen bond and a salt bridge to the carboxyl-moiety of lactate. Tyrosine 152 was suggested to form a hydrogen bond to the alpha-hydroxyl group and could be involved in stabilizing a transient carbanionic intermediate of the substrate. The tyrosine residues were replaced with phenylalanines (Y44F, Y152F), and arginine 293 was mutated to a lysine (R293K). In all cases catalysis was significantly decreased; however, the binding affinity for L-lactate did not decrease. Instead, the Kd measured for Y152F was 10-fold lower than that for the wild type enzyme. The products of turnover with Y152F were similar to those with wild type enzyme, with 70-80% of the reaction proceeding to form acetate, CO2, and H2O. The catalytic reactions with both Y44F and R293K were substantially uncoupled, with between 60 and 80% of the catalytic turnover forming pyruvate and H2O2. For all mutant forms the reoxidation of enzyme with oxygen in the absence of pyruvate occurred at a rate similar to that measured for the wild type enzyme. The most important effect of the mutations was in the ability to stabilize the transition state analog oxalate. A linear relationship was found between the rate of reduction of the enzyme flavin and the dissociation constant for the binding of oxalate, demonstrating that many individual residues contribute to the lowering of the energy of the transition state, in addition to specific functions being assignable to some specific residues.

Direct effect of protirelin (TRH) on PB[123I] in autonomous thyroid adenoma.

In a retrospective study of 27 cases of iodine deficiency and/or latent primary hypothyroidism and in 16 cases of thyroid adenoma with hyperthyreosis the routine radioiodine uptake test was combined with a protirelin (TRH) test. After TRH infusion, [PB*I] and TSH increased significantly in all of these 27 patients who served as controls for the hyperthyroid patients. At the same time, the conversion rate Q rose in 14 of the control patients, but it dropped in 13 cases, thus indicating a TSH-induced discharge from the thyroid of radioiodine containing substances that were not bound to serum proteins. In nine of the 16 patients with autonomous adenoma, PB[*I] rose slightly, but Q did not increase significantly. In seven of the 16 adenoma patients, both PB[*I] and Q even dropped slightly in the absence of measurable serum TSH, thus indicating a negative direct effect of TRH on thyroid hormone metabolism.