Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in vitro inactivation.

The oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase (ATase) is demonstrated in cell extracts of Bacillus subtilis. The rate of inactivation of ATase in vitro is apparently first order with respect to oxygen concentration and ATase activity. ATase inactivation in vitro (or in vivo) cannot be reactivated by a variety of reductants. ATase is significantly stabilized to oxygen-dependent inactivation in vitro in the presence of tetrasodium phosphoribosylpyrophosphate and glutamine together. The effects of the end product inhibitors, adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP), on the stability of ATase are antagonistic. AMP stabilizes ATase, whereas GMP destabilizes the enzyme. The stability of ATase can be manipulated over wide ranges by variations in the AMP/GM ratio. The effects of AMP and GMP on the inactivation of ATase in vitro are very specific. ATase is partially inhibited by 1,10-phenanthroline, suggesting that the enzyme contains iron (or some other chelatable metal ion). The inactivation of ATase in vitro is proposed to present a model for the reconstruction of the inactivation of ATase in stationary-phase cells of B. subtilis.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

Studies on in vivo inactivation of gramicidin S synthetase and its retardation

The oxygen-dependent in vivo inactivation of gramicidin S synthetase was investigated in Bacillus brevis ATCC 9999. Inhibitors of energy metabolism and of protein synthesis added to aerated cell suspensions did not provide any protection against inactivation, thus indicating that the process does not depend on energy-yielding metabolism nor on de novo protein synthesis. Organic thiols added to anaerobic long-term incubations retarded synthetase inactivation for several hours, whereas in short-term incubations of previously air-exposed cells they resulted in partial restoration of activity. The in vivo inactivation of the enzyme was found to be accompanied by a parallel drop in intracellular thiols. These results implicate enzyme SH oxidation as a mechanism of in vivo inactivation. Retardation of inactivation was achieved upon addition of utilizable carbon sources (glycerol, fructose, inositol) to aerated cell suspensions in buffer, the degree of stabilization being proportional to the ease of uptake and to the concentration of C source. This effect involves actual consumption of the exogenous C source and is accompanied by lower dissolved oxygen levels in the cell suspension. Pulsed additions of C source could retard inactivation but could not restore partly or fully lost activity. The C-source effect was blocked by the uncoupler dinitrophenol, while dissolved oxygen levels in the suspension remained low. C-source-supplemented cell suspensions incubated under air had a decreased intracellular redox state, as revealed by intracellular SH concentration.     

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands

The inactivation of glutamine phosphoribosylpyrophosphate amidotransferase by reaction of its iron-sulfur center with O2 is believed to be a physiologically important mode of regulation of this enzyme in Bacillus subtilis cells in the stationary phase of growth. Chemical and physical changes accompanying oxidation of the purified enzyme by O2 were studied. The iron of the 4Fe-4S center was oxidized to enzyme-bound high-spin Fe3+; the S2- was oxidized to a mixture of S0 bound as thiocystine and unidentified products. The oxidant appeared to be O2, rather than peroxide, superoxide, hydroxyl radical, or singlet oxygen. Gross physical changes in the oxidized enzyme were shown by its aggregation, decreased solubility, and altered circular dichroic spectrum. Experimental variables affecting the rate of oxidative inactivation were described; the most important of these was modulation of rates of inactivation by the allosteric inhibitors AMP, ADP, GMP, GDP and by the substrate P-Rib-PP. AMP was a potent stabilizer, whose effect was antagonized by P-Rib-PP. The other nucleotides, either acting singly or acting as synergistic pairs, were destabilizers and able to antagonize stabilization by AMP. The results are discussed in terms of the regulation of the stability of amidotransferase and its degradation in vivo.     

Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis.

Strains of Aspergillus nidulans carrying the orlA1 or tse6 allele are deficient in cell wall chitin and undergo lysis at restrictive temperatures. The strains are remediable by osmotic stabilizers or by the presence of N-acetylglucosamine (GlcNAc) in the medium. The remediation by GlcNAc suggests that the lesion(s) in chitin synthesis resides in the amino sugar biosynthetic pathway prior to the synthesis of N-acetylglucosamine-6-phosphate. orlA1 strains grown at permissive temperature exhibit an abnormally low specific activity for L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), the first enzyme unique to amino sugar synthesis. In addition, the enzyme produced is temperature sensitive in vitro. tsE6 strains grown at permissive temperature show virtually no amidotransferase activity. This finding is consistent with an extremely labile enzyme which is destroyed by cell breakage and extract preparation. The enzyme must be active in vivo at permissive temperatures since GlcNAc is not required for growth. Thus, two structural genes (orlA and tsE) are necessary for the amidotransferase activity. bimG11 strains are temperature sensitive for a type 1 protein phosphatase involved in cell cycle regulation and arrest in mitosis. Like orlA1 and tsE6 strains, conidia from bimG11 strains swell excessively when germinated and lyse; the germlings produced are deficient in chitin content. The amidotransferase from wild-type and mutant strains is sensitive to feedback inhibition by uridine diphosphate-N-acetylglucosamine. The sensitivity of the amidotransferase from bimG11 strains is dependent on growth temperature, while that from wild-type strains is independent of temperature. The enzyme can be desensitized in vitro under conditions consistent with a protein phosphatase reaction. It is proposed that amino sugar (and chitin biosynthesis) is partially regulated by phosphorylation-dephosphorylation of the amidotransferase or a protein regulator of the enzyme.     

Feedback inhibition of L-glutamine D-fructose 6-phosphate amidotransferase by uridine diphosphate N-acetylglucosamine in Neurospora crassa

The enzyme, l-glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa, which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l-glutamine or d-fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.     

Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.     

Differential stability of antigenic MHC class I-restricted synthetic peptides

Various synthetic peptides recognized as Ag by CTL in the context of MHC class I molecules were tested for stability in vitro and in vivo. Peptide inactivation in vitro was quantitated by titrating the amount of peptide required to sensitize target cells for lysis by specific CTL clones. The degree of inactivation after overnight incubation at 37 degrees C varied widely among a series of antigenic peptides. Some were nearly unaffected, whereas others lost activity by more than 100-fold or even 10,000-fold. However, no correlation was found between susceptibility to serum inactivation and antigenic potency as measured in short term cytolytic assays. No inactivation occurred at 4 degrees C, or at 37 degrees C in the absence of serum, under the conditions used. Serum inactivation most likely involved proteolysis because it could be inhibited by protease inhibitors. Moreover, presumed cleavage products of a radiolabeled susceptible peptide could be visualized by TLC. In vivo, the persistence of the antigenic activity of the injected peptides, either in extracellular fluids or on tumor target cells growing in an ascites form, correlated with the degree of stability found for the peptides in vitro. The differential stability of synthetic peptides may have important consequences for attempts to manipulate the development of an immune response in vivo.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.     

Growth on ethanol results in co-ordinated Saccharomyces cerevisiae response to inactivation of genes encoding superoxide dismutases

Superoxide dismutase (SOD) is an essential enzyme protecting cells against oxidative stress. However, its specific role under different conditions is not clear. To study the possible role of SOD in the cell during respiration, Saccharomyces cerevisiae single and double mutants with inactivated SOD1 and/or SOD2 genes growing on ethanol as an energy and carbon source were used. Activities of antioxidant and associated enzymes as well as the level of protein carbonyls were measured. SOD activity was significantly higher in a Mn-SOD deficient strain than that in the wild-type parental strain, but significantly lower in a Cu, Zn-SOD mutant. A strong positive correlation between SOD and catalase activities (R(2) = 0.99) shows possible protection of catalase by SOD from inactivation in vivo and/or decrease in catalase activity because of lower H(2)O(2) formation in the mutant cells. SOD deficiency resulted in a malate dehydrogenase activity increase, whereas glucose-6-phosphate dehydrogenase (G6PDH) activity was lower in SOD-deficient strains. Linear and non-linear positive correlations between SOD and isocitrate dehydrogenase activities are discussed. No changes in the activity of glutathione reductase and protein carbonyl levels support the idea that SOD-deficient cells are not exposed to strong oxidative stress during exponential growth of yeast cultures on ethanol.     

Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.     

Oxidative stress triggers thiol oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus aureus

The high-resolution two-dimensional protein gel electrophoresis technique combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to analyse the oxidative stress response in Staphylococcus aureus COL. Exponentially growing cells were supplemented with 100 mM H2O2 leading to a growth arrest lasting 30 min. The comparison of the two-dimensional pattern of cytoplasmic protein extracts of stressed and unstressed cells revealed only a few changes in the protein synthesis profile. However, the isoelectric points of Gap (glyceraldehyde-3-phosphate dehydrogenase), AhpC (alkylhydroperoxide reductase) and MvaS (HMG-CoA-synthase) changed strikingly. For analysis of the modification of Gap, tandem hybrid mass spectrometry (Q-Star) was used. The observed pI shift resulted from the oxidation to sulphonic acid of cysteine 151, which is crucial for catalytic activity. A drop in ATP and a complete inactivation of Gap was accompanied by the growth arrest. About 30 min after the addition of H2O2, the damaged Gap was still present, but a new protein spot at the original location became visible, representing the newly synthesized enzyme that is active again. This is accompanied by the restoration of Gap enzyme activity, ATP levels and recovery of growth. There is a strong correlation between growth, ATP level and Gap activity under oxidative stress conditions, indicating that the H2O2-triggered Gap inactivation might be one reason for growth arrest under these conditions. Our data indicate that the damaged Gap protein was not repaired.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Temperature-sensitive, osmotic-remedial mutants of Neurospora crassa: osmotic pressure induced alterations of enzyme stability

Three temperature-sensitive, adenine-requiring mutants of Neurospora crassa were found to be osmotic-remedial when non-penetrating solutes were used to increase the osmolarity of the growth medium. The affected enzyme (adenylosuccinase) from one of the mutants (ad-4, 44206t) was found to have higher levels of activity when the organism was grown at non-permissive temperatures under osmotic-remedial conditions than when it was grown with adenine as a nutritional supplement. The enzyme synthesized at 30 degrees C in the presence of adenine exhibited increased sensitivity to inhibition by high salt concentrations and a lowered stability toward heat denaturation, indicating that the remedial effect may be the result of changes in the physical properties of the enzyme molecule. Temperature shift experiments indicate that the enzyme which is synthesized at permissive temperatures or under osmotic-remedial conditions is also stable in vivo under non-permissive conditions. This suggests that the critical period for temperature sensitivity, and conversely osmotic remediability, may be during protein synthesis or during the conformational folding of the protein.     

Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from 2 mol of ATP, bicarbonate, and glutamine. CPS was inactivated by the glutamine analog, acivicin. In the presence of ATP and bicarbonate the second-order rate constant for the inactivation of the glutamine-dependent activities was 4.0 x 10(4) m(-1) s(-1). In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amidotransferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a conformational change within the amidotransferase domain of the small subunit that enhances the nucleophilic character of the thiol group required for glutamine hydrolysis. The acivicin-inhibited enzyme was crystallized, and the three-dimensional structure was determined by x-ray diffraction techniques. The thiol group of Cys-269 was covalently attached to the dihydroisoxazole ring of acivicin with the displacement of a chloride ion.     

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.     

Post-translational modification of a tryptophan biosynthetic enzyme. Precursor-product relationships in vivo and carbon-energy source dependence

Purified preparations of the bifunctional, tryptophan biosynthetic enzyme N-(5'-phosphoribosyl)anthranilate isomerase/indole-3-glycerol phosphate synthase of Escherichia coli K12 contain five forms of the protein (molecular mass = 49 kDa) which can be separated by isoelectric focusing or two-dimensional gel electrophoresis. The stability of the enzyme and its different forms was studied in exponentially growing, ammonium-starved and energy-depleted cultures using a dual-labeling, pulse-chase method. Labeled enzyme was isolated by standard purification techniques and by immunoprecipitation from crude extracts. The results demonstrated the following: 1) post-translational modification occurred in vivo, 2) modification occurred to the same extent in growing and ammonium-starved cells, 3) modification was dependent on a carbon-energy source for the cells, and 4) the enzyme was not degraded in growing or nongrowing cells. The pulse-chase data also indicated that one form of the protein (band 1) was the precursor of three other forms (bands 3-5) and that the relative amount of one form (band 2) remained constant.     

Oxidation of hypotaurine to taurine by ultraviolet irradiation.

Oxidation of hypotaurine to taurine is known to occur in vivo. Search for an enzyme performing that oxidation has been unsuccessful. However, fast and quantitative oxidation of hypotaurine (and other sulfinates) by ultraviolet irradiation has now been observed. The reaction is first order and pH-dependent, and its rate depends strongly on the kind of sulfinate irradiated. Only the corresponding sulfonate is recovered as the product under the relatively mild conditions used. Catalase or superoxide dismutase does not affect the oxidation, which is oxygen-dependent. A simple reaction scheme is proposed to account for the findings.     

Prolyl hydroxylase PHD3 activates oxygen-dependent protein aggregation

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.