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S D Aust

Publications and source records attributed to S D Aust.

At least 73 records · Page 4Linked to original sources
PubMed1995

Lignin peroxidases can also oxidize manganese.

The peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium include lignin peroxidases and manganese-dependent peroxidases. The major isozymes, called lignin peroxidases, are thought to oxidize chemicals directly. The manganese-dependent peroxidases (H3, H4, H5, and H9) are relatively minor, making up only a fraction of the total peroxidase protein. However, we have found that lignin peroxidases will also catalyze the H2O2-dependent oxidation of Mn2+ to Mn3+. We have used lignin peroxidase isozyme H2 (LiPH2) to characterize the manganese peroxidase activity of lignin peroxidases. Transient state kinetic studies were used to obtain a second-order rate constant of 4.2 x 10(4) M-1 S-1 for the reaction of LiPH2-compound I with free or chelated Mn2+ at pH 6.0. This reaction was too fast to monitor at pH 4.5. Only chelated Mn2+ could reduce LiPH2-compound II to ferric enzyme. The Mn(2+)-chelate (oxalate) first bound LiPH2-compound II with a Kd of (1.5 +/- 0.3) x 10(-5) M and then reduced LiPH2-compound II to ferric enzyme with a first order rate constant of 215 +/- 6 S-1. Steady-state kinetic studies on LiPH2 were performed by directly monitoring the formation of Mn(3+)-oxalate. These results show that oxidation of Mn2+ by a lignin peroxidase does not occur through free radical mediation as proposed previously [Popp et al. (1990) Biochemistry 29, 10475-10480). Electron spin resonance and oxygen evolution studies also indicate that Mn2+ is directly oxidized by LiPH2.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1995

Evidence for formation of the veratryl alcohol cation radical by lignin peroxidase.

Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxidation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-electron oxidized intermediate, the veratryl alcohol cation radical (VA.+). The ESR spectrum of VA.+ was first obtained in a fast-flow system with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. This ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by computer simulation of the ESR spectrum and calculation of spin and charge densities on the radical. An identical radical signal was observed with LiP, also in a fast-flow incubation containing 10 microM LiP, 2 mM VA, and 500 microM H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP were due to the same radical species. The VA.+ had a distinct visible spectrum in 98% H2SO4 with an absorbance maximum at 529 nm. The extinction coefficient of the VA.+ spectral band at 529 nm was calculated to be 11,000 M-1 cm-1. The VA.+ was found to be a strong acid, as are other cation radicals, with the pKa at -1.0 pH. This value was determined by quantitating both the concentration of VA.+ by visual and ESR spectrometry and the g-value of the ESR signal at various pH values.

Benzyl Alcohols↗
PubMed1995

Evidence for veratryl alcohol as a redox mediator in lignin peroxidase-catalyzed oxidation.

We have examined the hypothesis that veratryl alcohol (VA) may act as a redox mediator in lignin peroxidase (LiP)-catalyzed oxidations. The oxidation of chlorpromazine (CPZ) by this system was used to evaluate this hypothesis. Chlorpromazine can be oxidized by one electron to form a stable cation radical (CPZ.+). This cation radical can be oxidized by another electron to the sulfoxide (CPZSO). These oxidation steps are easily monitored, making CPZ a useful chemical to investigate redox mediation by VA. Lignin peroxidase oxidized CPZ to CPZ.+ whether or not VA was present. The inclusion of VA, however, stimulated CPZ oxidation to CPZ.+ and subsequent oxidation of CPZ.+ to CPZSO. In the absence of VA, the initial rates of CPZ oxidation by LiP were CPZ concentration-dependent. However, when saturating concentrations of VA were added, the oxidation of CPZ and CPZ.+ became independent of CPZ concentration. When the oxidation of VA to veratryl aldehyde was examined, increasing concentrations of CPZ produced a lag in veratryl aldehyde appearance proportional to the concentration of CPZ. Conversely, increasing concentrations of VA never inhibited CPZ oxidation. Transient-state kinetic studies indicated that both VA and CPZ reduced the compound I and compound II forms of LiP. However, when saturating concentrations of VA were utilized, LiP turnover was independent of CPZ concentration. We suggest these data demonstrate that VA may act as a redox mediator for the indirect oxidation of compounds by LiP.

Benzyl Alcohols↗
PubMed1995

Hydroxylation of deoxyguanosine in DNA by copper and thiols.

DNA was incubated with glutathione (GSH) and copper and then assayed for 8-hydroxydeoxyguanosine (8-OHdG) in order to better understand the antioxidant and prooxidant characteristics of GSH in copper-dependent DNA damage. Ratios of GSH to Cu(II) less than 3 resulted in 8-OHdG production; however, higher ratios did not generate 8-OHdG. A combination of GSH and Cu(I) (10:1) was used to determine if DNA oxidation occurred upon the addition of H2O2. No increase in 8-OHdG was noted until the concentration of H2O2 was almost half that of GSH, and then a substantial increase of 8-OHdG was detected. The stoichiometry of thiol oxidation by H2O2 was 2 mol GSH oxidized per 1 mol H2O2. Oxidation of Cu(I) was not detected until most of the thiol had been oxidized. When cysteine and Cu(I) was used instead of GSH and Cu(I), there was considerable hydroxylation of deoxyguanosine. The glycyl carboxyl, the gamma-glutamate carboxyl, and the amine of GSH were altered to determine their role in the peptide's ability to inhibit Cu-dependent damage. In the presence of Cu(I), H2O2, and DNA, these GSH analogs behaved similarly to GSH. However, when S-methylglutathione was used in this system, it was very effective at promoting oxidative damage to DNA. This indicated that the thiol ligand of GSH was essential for inhibition of Cu-dependent damage, while the carboxyl groups and the amine were not essential ligands. In conclusion, GSH can catalyze the in vitro hydroxylation of deoxyguanosine when the ratio of GSH to Cu is low, however, when the ratio is high GSH is an effective antioxidant.

Copper↗
PubMed1995

Veratryl alcohol-mediated indirect oxidation of phenol by lignin peroxidase.

Veratryl alcohol (VA) oxidation by lignin peroxidase (LiP) was inhibited by phenol. The enzyme was quickly converted to compound III, an inactive intermediate. However, as soon as VA began to be oxidized, compound II was observed. The lag period before VA oxidation was affected by the concentrations of both phenol and VA. The addition of VA increased the extent of phenol oxidation and the kinetics of phenol oxidation in the presence of VA were similar to those of VA oxidation. Previously it was shown that the VA cation radical (VA.+) was responsible for the conversion of compound III back to ferric enzyme [D. P. Barr and S. D. Aust (1994) Arch. Biochem. Biophys. 312, 511-515]. Here we observed that the reversion of compound III to active ferric enzyme in the presence of VA was prevented by addition of phenol, suggesting that VA.+ oxidizes phenol to phenoxyl radical. This hypothesis was also supported by the observation that O2 consumption during VA oxidation was inhibited by the addition of phenol. All of these results suggested that VA was first oxidized by LiP to VA.+ and then it oxidized phenol to phenoxyl radical while VA.+ was reduced back to VA. Activity was lost as compound III accumulated since the VA.+ was consumed by the oxidation of phenol. After all of the phenol was oxidized, VA.+ became available to convert compound III back to ferric enzyme.

Benzyl Alcohols↗
PubMed1995

Inactivation of lignin peroxidase by hydrogen peroxide during the oxidation of phenols.

The oxidation of phenols by lignin peroxidase (LiP) was characterized by a rapid decrease in enzyme activity. The initial oxidation rate of phenol decreased at moderately high (i.e., 400 microM) concentrations of H2O2. Similar results were obtained with several phenols. However, LiP was not inactivated during the oxidation of veratryl alcohol (VA). The apparent second-order rate constants for the reaction of compound II with phenol and VA at pH 3.5 were very similar, about 2.1 x 10(4) and 4.7 x 10(4) M-1 s-1, respectively. However, unlike VA, phenols could not convert compound III back to ferric enzyme. The visible absorption spectra of compound II and III were observed during oxidation of VA and phenols, respectively. These results suggest that the inactivation of LiP during the oxidation of phenols was mainly due to the accumulation of compound III, which was attributed to the inability of phenols or phenoxyl radicals to revert compound III to ferric enzyme. All of these results also suggested that the inactivation mechanism of LiP during oxidation of phenols was different from that during oxidation of anisyl alcohol, since anisyl alcohol was neither a substrate of compound II nor could it revert compound III [K. Valli et al. (1990) Biochemistry 29, 8535-8539 and R.S. Koduri and M. Tien (1994) Biochemistry 33, 4225-4230].

Benzyl Alcohols↗
PubMed1995

Impact of nutrients on cellular lipid peroxidation and antioxidant defense system.

A symposium entitled Impact of Nutrients on Cellular Lipid Peroxidation and Antioxidant Defense System was held at the 33rd Annual Meeting of the Society of Toxicology (SOT) at the Loews Anatole Hotel in Dallas, Texas. The symposium was sponsored by the Food Safety Specialty Section and focused on the role of particular nutrients in cellular lipid peroxidation and antioxidant defense system. Emphasis was placed on defining underlying mechanisms for damage and protection, as well as potential ramification in human health issues. The following are extractions of some of the highlights from each presentation.

Antioxidants↗
PubMed1995

Ferritin as a source of iron and protection from iron-induced toxicities.

The iron storage protein ferritin can contribute to or protect against toxicities which involve iron. Iron can catalyze the oxidation of lipid, protein, DNA and various biomolecules that can reduce iron. Iron can be reduced and released from ferritin by the free radical form of various toxins or superoxide resulting from oxygen reduction by chemicals which redox cycle. Iron can also increase ferritin synthesis by an iron-binding protein which releases from an iron-responsive element in mRNA for ferritin. This increase in ferritin synthesis provides a non-reactive storage site for iron. The mechanism by which iron is placed into ferritin is unknown. We propose that it is catalyzed by ceruloplasmin, the copper-containing ferroxidase that loads iron into transferrin. We believe that the ferroxidase activity, thought to reside in the heavy chain of ferritin, is an artifact resulting from ferrous iron autoxidation. We load iron into ferritin with ceruloplasmin so ferritin plus ceruloplasmin is an effective 'antioxidant'.

Animals↗
PubMed1995

Mechanisms of degradation by white rot fungi.

White rot fungi use a variety of mechanisms to accomplish the complete degradation of lignin and a wide variety of environmental pollutants. Both oxidative and reductive reactions are required for the metabolism of both lignin and environmental pollutants. The fungi secrete a family of peroxidases to catalyze both direct and indirect oxidation of chemicals. The peroxidases can also catalyze reductions using electron donors to generate reductive radicals. A cell-surface membrane potential can also be used to reduce chemicals such as TNT.

Basidiomycota↗
PubMed1994

Oxalate-dependent reductive activity of manganese peroxidase from Phanerochaete chrysosporium.

The mechanism of oxalate-dependent reductive activity of a manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing Mn-peroxidase, Mn2+, oxalate, H2O2, ferric chloride, and 1,10-phenanthroline. Only catalytic amounts of H2O2 were required. Oxygen consumption was also observed in reaction mixtures containing Mn-peroxidase, Mn2+, oxalate, and H2O2 and was inhibited by the addition of ferric iron. Electron spin resonance studies, using the spin traps 5,5-dimethyl-1-pyrroline-N-oxide and alpha-4-pyridyl-1-oxide-N-t-butylnitrone were used to obtain evidence for the production of the formate radical (CO2.-) and superoxide (O2.-) in a reaction mixture containing Mn2+, oxalate and H2O2. It was concluded that both CO2.- (anaerobic conditions) and O2.- (aerobic conditions) could reduce ferric iron. The dismutation of some O2.- would produce H2O2 to provide a constant supply of H2O2.

Aerobiosis↗
PubMed1994

Conversion of lignin peroxidase compound III to active enzyme by cation radicals.

It has been previously reported that the catalytically inactive compound III form of lignin peroxidase is formed during the oxidation of certain chemicals such as phenols (P.J. Harvey and J.M. Palmer, 1990, J. Biotechnol. 13, 169-179). Here we provide evidence that the cation radicals of methoxybenzenes such as 1,2,4,5-tetramethoxybenzene (TMB) and veratryl alcohol promote the oxidative conversion of compound III back to ferric enzyme. Two kinetic phases were observed during the oxidation of TMB by lignin peroxidase. In the first phase the formation of TMB cation radical and compound III were observed simultaneously. The second phase involved a rapid disappearance of compound III and the TMB cation radical. Ferric enzyme appeared concomitantly with the disappearance of compound III. The TMB cation radical, generated electrochemically, was able to convert compound III to ferric enzyme. Comparative studies using veratryl alcohol were performed and supported the idea that the cation radical of these chemicals are capable of reactivating compound III. The significance of these reactions with respect to lignin peroxidase catalysis are discussed.

Anisoles↗
PubMed1994

Transcription of ligninase H8 by Phanerochaete chrysosporium under nutrient nitrogen sufficient conditions.

Dot blot analyses showed that more ligninase H8 mRNA was present in ammonium sufficient cultures of Phanerochaete chrysosporium than in ammonium limited cultures. Reverse transcription followed by polymerase chain reaction and DNA sequencing verified that H8 mRNA was present under both conditions. Fast protein liquid chromatography profiles indicated that H8 was present but lacked heme in ammonium sufficient cultures. These data indicated that H8 was transcribed and translated by day 5 under ammonium sufficient conditions, but was inactive due to lack of heme.

Agaricales↗
PubMed1994

Effect of superoxide and superoxide dismutase on lignin peroxidase-catalyzed veratryl alcohol oxidation.

We have shown that superoxide (O2.-) is produced during the oxidation of veratryl alcohol by lignin peroxidase (LiP) by the reaction of the veratryl alcohol cation radical with hydrogen peroxide (D. B. Barr, M. M. Shah, and S. D. Aust, 1993, J. Biol. Chem. 268, 241-244). Compound III, an inactive form of peroxidases can be formed by reaction of the ferric enzyme with O2.-. We therefore studied the effects of O2.- and superoxide dismutase (SOD) on the veratryl alcohol oxidase activity of LiP. SOD enhanced the rate of veratryl alcohol oxidation by LiP and veratryl alcohol oxidation was inhibited by the addition of KO2. Upon the addition of KO2, activity was also preceded by a lag period. Under steady-state turnover conditions (i.e., for veratryl alcohol oxidation), the addition of KO2 resulted in the formation of LiP compound III. Compound II of LiP was observed following a time period that correlated with the lag prior to veratryl aldehyde formation. The extent of the lag preceding veratryl aldehyde formation increased with increasing concentrations of KO2 and decreased with increasing concentrations of veratryl alcohol. It was postulated that during the lag period the veratryl alcohol cation radical reacted with compound III to regenerate the native enzyme. In this process the veratryl alcohol cation radical was reduced to veratryl alcohol, and thus, no veratryl aldehyde was detected during the lag period.

Agaricales↗
PubMed1994

Thiol-mediated NTA-Fe(III) reduction and lipid peroxidation.

The nephrotoxicity of nitrilotriacetate chelated Fe(III) (NTA-Fe(III)) has been linked to the metabolism of glutathione (GSH) by gamma-glutamyl transpeptidase and a dipeptidase. The products of these enzymes are cysteinyl-glycine (cys-gly) and cysteine (cys), which are proposed to be the reductants of NTA-Fe(III) to cause oxidative damage to various biomolecules. The ability of cys-gly and cys to cause in vitro NTA-Fe(III)-dependent lipid peroxidation correlated directly with their ability to reduce NTA-Fe(III). GSH reduced iron at a much slower rate and did not stimulate lipid peroxidation. It has been proposed that GSH, cys-gly and cys reduce iron at different rates because their thiols have different pKas. However, increasing the amount of GS-, by raising the pH, did not cause a corresponding increase in the rate of iron reduction. The monomethyl ester of GSH reduced NTA-Fe(III) at the same rate as GSH, but the dimethyl ester of GSH reduced NTA-Fe(III) approximately 30 times faster. From this we conclude that GSH does not reduce NTA-Fe(III) at the same rate as cys-gly and cys because of the liganding between GSH and the iron.

Carcinogens↗
PubMed1994

The role of oxalate in lignin peroxidase-catalyzed reduction: protection from compound III accumulation.

Reduction may be an important step in the degradation of some highly oxidized environmental pollutants by Phanerochaete chrysosporium. Lignin peroxidases (LiP) from P. chrysosporium are able to catalyze reductive reactions using veratryl alcohol (VA) as a mediator and either oxalate or EDTA as electron donors. Reduction of oxygen to superoxide, monitored by oxygen consumption, was used as a measure of the reductive activity of LiP. In the presence of EDTA, the rate of O2 reduction catalyzed by LiP decreased with time and increasing concentrations of H2O2. When oxalate replaced EDTA, LiP-catalyzed O2 reduction did not decrease with time, and increasing concentrations of H2O2 increased the duration and extent of O2 reduction. LiP was converted to the compound III state in the presence of EDTA, H2O2, and veratryl alcohol. When oxalate replaced EDTA, compound II was observed. The importance of the veratryl alcohol cation radical (VA.+) in the conversion of LiP compound III to active enzyme has been previously examined (D.P. Barr and S.D. Aust, 1994, Arch. Biochem. Biophys. 311, 378-382). We propose that rapid reduction of VA.+ by EDTA results in accumulation of LiP compound III and the loss of activity resulting in a decrease in LiP-catalyzed reduction reactions. Oxalate is less effective in reducing the VA.+, therefore, some VA.+ remains to convert compound III to active enzyme and maintain LiP-catalyzed reduction reactions. Thus oxalate, a normal secondary metabolite of P. chrysosporium, is a suitable candidate for mediating reduction reactions by LiP in vivo.

Edetic Acid↗
PubMed1994

Pollutant degradation by white rot fungi.

The white rot fungi technology is very different from other more well-established methods of bioremediation (e.g., bacterial systems). The differences are primarily due to the mechanisms discussed previously. The unusual mechanisms used by the fungi provide them with several advantages for pollutant degradation, but the complexity of these mechanisms has also made the technology slow to emerge as a viable method of bioremediation. One distinct advantage that white rot fungi have over bacterial systems is that they do not require preconditioning to a particular pollutant. Bacteria must be preexposed to a pollutant to allow the enzymes that degrade the pollutant to be induced. The pollutant must also be present in a significant concentration, otherwise induction of enzyme synthesis will not occur. Therefore, there is a finite level to which pollutants can be degraded by bacteria. In contrast, the degradative enzymes of white rot fungi are induced by nutrient limitation. Thus, cultivate the fungus on a nutrient that is limited in something, and the degradative process will be initiated. Also, because the induction of the lignin-degrading system is not dependent on the chemical, pollutants are degraded to near-nondetectable levels by white rot fungi. Another unique feature of pollutant degradation by white rot fungi involves kinetics. The process of chemical conversion by these fungi occurs via a free-radical process, and thus the degradation of chemicals often follows pseudo-first-order kinetics. In fact, in several studies, it has been found that the rate of mineralization or disappearance of a pollutant is proportional to the concentration of the pollutant. This makes the time required to achieve decontamination more important than the rate of degradation. Because the metabolism of chemicals by bacteria involves mostly enzymatic conversions, pollutant degradation often follows Michaelis-Menton-type kinetics. Therefore, Km values of various degradative enzymes with respect to the pollutant must be considered when using bacteria for bioremediation. Considering this, the solubility of a pollutant or a mixture of pollutants might also present a problem for bacterial degradation. In contrast, using a nonspecific free-radical-based mechanism, the fungi are able to degrade insoluble complex mixtures of pollutants, such as creosote (Aust and Bumpus 1989) and Arochlor (Bumpus and Aust 1987b). Inexpensive nutrient sources, such as sawdust, wood chips, surplus grains, and agricultural wastes, can be used to effectively cultivate white rot fungi.(ABSTRACT TRUNCATED AT 400 WORDS)

Basidiomycota↗
PubMed1994

Detection of Phanerochaete chrysosporium in soil by PCR and restriction enzyme analysis.

A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA. Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 micrograms of total DNA extracted from 1 g of soil.

Base Sequence↗