Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16.

The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation. Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2. The enzyme could also be partly activated either with NADH alone or with H2 alone. The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone. Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration. The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction. Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity. The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase.     

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.     

L-Methionine-SR-sulfoximine as a probe for the role of glutamine synthetase in nitrogenase switch-off by ammonia and glutamine in Rhodopseudomonas palustris

Methionine sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase of Rhodopseudomonas palustris restored nitrogenase activity to cells in which nitrogenase had been completely inhibited by ammonia switch-off. After addition of MSX, there was a lag period before nitrogenase activity was fully restored. During this lag, glutamine synthetase activity progressively decreased, and near the time of its complete inhibition, nitrogenase activity resumed. Nitrogenase switch-off by ammonia thus required active glutamine synthetase. Glutamine itself caused nitrogenase inhibition whose reversal by MSX depended on the relative ratio of MSX to glutamine. Unlike ammonia, glutamine inhibited nitrogenase under conditions where glutamine synthetase activity was absent. This indicates that glutamine is the effector molecule in nitrogenase switch-off, for instance by interacting with the enzymatic system for Fe protein inactivation. The effects of glutamine and MSX were also dependent on the culture age. Possible explanation for this and for the competitive effects are a common binding site within the regulatory apparatus for nitrogenase, or, in part, within a common transport system. Some observations with MSX were extended to Rhodopseudomonas capsulata and agreed with those in R. palustris.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Natural occurrence of the mycotoxin penitrem a in moldy cream cheese

The occurrence of the mycotoxin penitrem A in refrigerated cream cheese that was involved in the intoxication of two dogs is described. Penitrem A and the fungus Penicillium crustosum were isolated from the cream cheese and identified. This is believed to be the first definitive case of the natural occurrence of penitrem A.     

Nitrogenase switch-off by ammonia in Rhodopseudomonas palustris: Loss under nitrogen deficiency and independence from the adenylylation state of glutamine synthetase

Nitrogenase activity in Rhodopseudomonas palustris is subject to a rapid switch-off in response to exogenous ammonia. When cells were grown on limiting nitrogen and eventually became nitrogen deficient, nitrogenase synthesis was fully derepressed but the enzyme was insensitive to ammonia. The transformation of ammonia-sensitive to ammonia-insensitive cells was a slow, but fully reversible process. The switch-off effect in ammonia-sensitive cells paralleled changes in the adenylylation state of glutamine synthetase. Ammonia-insensitive cells, however, showed similar changes in glutamine synthetase activity although nitrogenase activity was unaffected. We conclude that nitrogenase regulation and adenylylation of glutamine synthetase are independent processes, at least under conditions of nitrogen deficiency.     

Inhibition, Inactivation, and Recovery of Ammonia-Oxidizing Activity in Cometabolism of Trichloroethylene by Nitrosomonas europaea

The kinetics of the cometabolism of trichloroethylene (TCE) by the ammonia-oxidizing soil bacterium Nitrosomonas europaea in short-term (<10-min) incubations were investigated. Three individual effects of TCE cometabolism on this bacterium were characterized. First, we observed that TCE is a potent competitive inhibitor of ammonia oxidation by N. europaea. The K(infi) value for TCE (30 (mu)M) is similar to the K(infm) for ammonia (40 (mu)M). Second, we examined the toxicity associated with TCE cometabolism by N. europaea. Stationary-phase cells of N. europaea oxidized approximately 60 nmol of TCE per mg of protein before ammonia-oxidizing activity was completely inactivated by reactive intermediates generated during TCE oxidation. At the TCE concentrations used in these experiments, ammonia did not provide significant protection against inactivation. Third, we have determined the ability of cells to recover ammonia-oxidizing activity after exposure to TCE. Cells recovering from TCE inactivation were compared with cells recovering from the specific inactivation of ammonia-oxidizing activity by light. The recovery kinetics were indistinguishable when 40% or less of the activity was inactivated. However, at increased levels of inactivation, TCE-inactivated cells did not recover as rapidly as light-inactivated cells. The kinetics of recovery appear to be dependent on both the extent of inactivation of ammonia-oxidizing activity and the degree of specificity of the inactivating treatment.     

Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response

Pure cultures of ammonia-oxidizing bacteria, Nitrosomonas europaea, were exposed to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), chloroform (CF), 1,2-dichloroethane (1,2-DCA), or carbon tetrachloride (CT), in the presence of ammonia, in a quasi-steady-state bioreactor. Estimates of enzyme kinetics constants, solvent inactivation constants, and culture recovery constants were obtained by simultaneously fitting three model curves to experimental data using nonlinear optimization techniques and an enzyme kinetics model, referred to as the inhibition, inactivation, and recovery (IIR) model, that accounts for inhibition of ammonia oxidation by the solvent, enzyme inactivation by solvent product toxicity, and respondent synthesis of new enzyme (recovery). Results showed relative enzyme affinities for ammonia monooxygenase (AMO) of 1,1-DCE approximately TCE > CT > NH(3) > CF > 1,2-DCA. Relative maximum specific substrate transformation rates were NH(3) > 1,2-DCA > CF > TCE approximately 1,1-DCE > CT (=0). The TCE, CF, and 1,1-DCE inactivated the cells, with 1,1-DCE being about three times more potent than TCE or CF. Under the conditions of these experiments, inactivating injuries caused by TCE and 1,1-DCE appeared limited primarily to the AMO enzyme, but injuries caused by CF appeared to be more generalized. The CT was not oxidized by N. europaea while 1,2-DCA was oxidized quite readily and showed no inactivation effects. Recovery capabilities were demonstrated with all solvents except CF. A method for estimating protein yield, the relationship between the transformation capacity model and the IIR model, and a condition necessary for sustainable cometabolic treatment of inactivating substrates are presented. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 520-534, 1997.     

Regulation of the Synthesis and Activity of Ammonia Monooxygenase in Nitrosomonas europaea by Altering pH To Affect NH(inf3) Availability

The obligately ammonia-oxidizing bacterium Nitrosomonas europaea was incubated in medium containing 50 mM ammonium. Changes in the concentration of nitrite, the pH, and the NH(inf4)(sup+)- and NH(inf2)OH-dependent O(inf2) uptake activities of the cell suspension were monitored. The NH(inf4)(sup+)-dependent O(inf2) uptake activity doubled over the first 3 h of incubation and then slowly returned to its original level over the following 5 h. The extent of stimulation of NH(inf4)(sup+)-dependent O(inf2) uptake activity was decreased by lowering the initial pH of the medium. Radiolabeling studies demonstrated that the stimulation of NH(inf4)(sup+)-dependent O(inf2) uptake activity involved de novo synthesis of several polypeptides. Under O(inf2)-limited conditions, the stimulated NH(inf4)(sup+)-dependent O(inf2) uptake activity was stabilized. Rapid, controlled, and predictable changes in this activity could be caused by acidification of the medium in the absence of ammonia oxidation. These results indicate that the NH(inf4)(sup+)-dependent O(inf2) uptake activity in N. europaea is strongly regulated in response to NH(inf3) concentration.     

Chloroform Cometabolism by Butane-Grown CF8, Pseudomonas butanovora, and Mycobacterium vaccae JOB5 and Methane-Grown Methylosinus trichosporium OB3b

Chloroform (CF) degradation by a butane-grown enrichment culture, CF8, was compared to that by butane-grown Pseudomonas butanovora and Mycobacterium vaccae JOB5 and to that by a known CF degrader, Methylosinus trichosporium OB3b. All three butane-grown bacteria were able to degrade CF at rates comparable to that of M. trichosporium. CF degradation by all four bacteria required O(inf2). Butane inhibited CF degradation by the butane-grown bacteria, suggesting that butane monooxygenase is responsible for CF degradation. P. butanovora required exogenous reductant to degrade CF, while CF8 and M. vaccae utilized endogenous reductants. Prolonged incubation with CF resulted in decreased CF degradation. CF8 and P. butanovora were more sensitive to CF than either M. trichosporium or M. vaccae. CF degradation by all three butane-grown bacteria was inactivated by acetylene, which is a mechanism-based inhibitor for several monooxygenases. Butane protected all three butane-grown bacteria from inactivation by acetylene, which indicates that the same monooxygenase is responsible for both CF and butane oxidation. CF8 and P. butanovora were able to degrade other chlorinated hydrocarbons, including trichloroethylene, 1,2-cis-dichloroethylene, and vinyl chloride. In addition, CF8 degraded 1,1,2-trichloroethane. The results indicate the potential of butane-grown bacteria for chlorinated hydrocarbon transformation.