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M Tien

Publications and source records attributed to M Tien.

At least 73 records · Page 4Linked to original sources
PubMed1988

Structure and regulation of a lignin peroxidase gene from Phanerochaete chrysosporium.

We have been investigating the structure and regulation of the lignin peroxidase genes from Phanerochaete chrysosporium. The gene for lignin peroxidase isozyme H8 was isolated and sequenced. The gene is split into nine exons and eight introns. The introns have consensus sequences both at the splice junctions and at an internal element which may be involved in the splicing mechanism. The promoter contains the eukaryotic consensus elements: a TATA box at -78 and a ACAAT box at -106. Transcription initiates downstream from these sequences as verified by S1 nuclease mapping. Induction of the genes for the lignin peroxidase isozymes during growth under nitrogen and under carbon limitation were compared by analyses of RNA and protein. The results indicate that the lignin peroxidase isozymes are differentially regulated in response to environmental stress.

Base Sequence↗
PubMed1988

Studies on compound I formation of the lignin peroxidase from Phanerochaete chrysosporium.

Ligninase, isolated from the wood-destroying fungus Phanerochaete chrysosporium, catalyzes the oxidation of lignin and lignin-related compounds. Ligninase reacts with H2O2 to form the classical peroxidase intermediates Compounds I and II. We have determined the activation energy of ligninase Compound I formation to be 5.9 kcal/mol. The effect of pH and ionic strength on the rate of ligninase Compound I formation was studied. In contrast to all other peroxidases, no pH effect was observed. This is despite homology of active-site amino acids residues (Tien, M., and Tu, C.-P. D. (1987) Nature 326, 520-523) which are proposed to affect the pH profile of Compound I formation. Ligninase Compound I formation can also be supported by organic peroxides. The second-order rate constants with the organic peroxides are lower, suggesting that H2O2 is the preferred substrate.

Amino Acid Sequence↗
PubMed1988

Oxidation of Thianthrene by the Ligninase of Phanerochaete chrysosporium.

The oxidation of heterocyclic sulfur compounds reported to be part of the macrostructure of coal and petroleum was investigated. The oxidation of thianthrene solubilized in 10% dimethylformamide to thianthrene monosulfoxide in the presence of hydrogen peroxide was catalyzed by the ligninase from Phanerochaete chrysosporium.

Journal Article↗
PubMed1987

Selection and improvement of lignin-degrading microorganisms: potential strategy based on lignin model-amino Acid adducts.

The purpose of this investigation was to test a potential strategy for the ligninase-dependent selection of lignin-degrading microorganisms. The strategy involves covalently bonding amino acids to lignin model compounds in such a way that ligninase-catalyzed cleavage of the models releases the amino acids for growth nitrogen. Here we describe the synthesis of glycine-N-2-(3,4-dimethoxyphenyl)ethane-2-ol (I) and demonstrate that growth (as measured by mycelial nitrogen content) of the known lignin-degrading basidiomycete Phanerochaete chrysosporium Burds. with compound I as the nitrogen source depends on its production of ligninase. Ligninase is shown to catalyze the oxidative C-C cleavage of compound I, releasing glycine, formaldehyde, and veratraldehyde at a 1:1:1 stoichiometry. P. chrysosporium utilizes compound I as a nitrogen source, but only after the cultures enter secondary metabolism (day 3 of growth), at which time the ligninase and the other components of the ligninolytic system (lignin --> CO(2)) are expressed. Compound I and related adducts have potential not only in the isolation of lignin-degrading microbes but, perhaps of equal importance, in strain improvement.

Journal Article↗
PubMed1987

Properties of ligninase from Phanerochaete chrysosporium and their possible applications.

The wood-degrading fungus Phanerochaete chrysosporium Burds produces a family of enzymes which degrade lignin and lignin-like substrates. These ligninases exhibit a high degree of homology in being hemeprotein peroxidases, in Mr, in cross reactivity to polyclonal antibodies, in being glycosylated, and in catalytic properties. The predominant ligninase is able to generate cation radicals in its aromatic substrates. These radicals can undergo a variety of reactions thus explaining the nonspecific nature of the enzyme. A similar mechanism is suggested for the other isoenzymes. There are numerous potential applications for ligninases. These include: biopulping, waste treatment of byproduct lignins, detoxification of environmental pollutants, and modification of lignins to produce small molecular weight organics.

Basidiomycota↗
PubMed1986

Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol beta-aryl ether substructure of lignin.

This study examined the ligninase-catalysed degradation of lignin model compounds representing the arylglycerol beta-aryl ether substructure, which is the dominant one in the lignin polymer. Three dimeric model compounds were used, all methoxylated in the 3- and 4-positions of the arylglycerol ring (ring A) and having various substituents in the beta-ether-linked aromatic ring (ring B), so that competing reactions involving both rings could be compared. Studies of the products formed and the time courses of their formation showed that these model compounds are oxidized by ligninase (+ H2O2 + O2) in both ring A and ring B. The major consequence with all three model compounds is oxidation of ring A, leading primarily to cleavage between C(alpha) and C(beta) (C(alpha) being proximal to ring A), and to a lesser extent to the oxidation of the C(alpha)-hydroxy group to a carbonyl group. Such C(alpha)-oxidation deactivates ring A, leaving only ring B for attack. Studies with C(alpha)-carbonyl model compounds corresponding to the three basic model compounds revealed that oxidation of ring B leads in part to dealkoxylations (i.e. to cleavage of the glycerol beta-aryl ether bond and to demethoxylations), but that these are minor reactions in the model compounds most closely related to lignin. Evidence is also given that another consequence of oxidation of ring B in the C(alpha)-carbonyl model compounds is formation of unstable cyclohexadienone ketals, which can decompose with elimination of the beta-ether-linked aromatic ring. The mechanisms proposed for the observed reactions involve initial formation of aryl cation radicals in either ring A or ring B. The cation radical intermediate from one of the C(alpha)-carbonyl model compounds was identified by e.s.r. spectroscopy. The mechanisms are based on earlier studies showing that ligninase acts by oxidizing appropriately substituted aromatic nuclei to aryl cation radicals [Kersten, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 2609-2612; Hammel, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 8348-8353].

Binding Sites↗
PubMed1986

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds.

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.

Basidiomycota↗
PubMed1985

Mechanism of oxidative C alpha-C beta cleavage of a lignin model dimer by Phanerochaete chrysosporium ligninase. Stoichiometry and involvement of free radicals.

The hemoprotein ligninase of Phanerochaete chrysosporium Burds. catalyzes the oxidative cleavage of lignin model dimers between C alpha and C beta of their propyl side chains. The model dimers hitherto used give multiple products and complex stoichiometries upon enzymatic oxidation. Here we present experiments with a new model dimer, 1-(3,4-dimethoxyphenyl)-2-phenylethanediol (dimethoxyhydrobenzoin, DMHB) which is quantitatively cleaved by ligninase in air to give benzaldehyde and veratraldehyde according to the stoichiometry: 2DMHB + O2----2PhCHO + 2Ph(OMe)2CHO. Catalytic amounts of H2O2 are required for this aerobic reaction. Under anaerobic conditions, ligninase uses H2O2 as the oxidant for cleavage: DMHB + H2O2----PhCHO + Ph(OMe)2CHO. Electron spin resonance experiments done in the presence of spin traps, 2-methyl-2-nitrosopropane or 5,5-dimethyl-1-pyrroline-N-oxide, show that C alpha-C beta cleavage yields alpha-hydroxybenzyl radicals as intermediate products. Under anaerobic conditions, these radicals react further to give the final aldehyde products. In air, O2 adds to the carbon-centered radicals, probably giving alpha-hydroxybenzylperoxyl radicals which fragment to yield superoxide, benzaldehyde, and veratraldehyde. These results lead us to propose a mechanism for C alpha-C beta cleavage in which attack by ligninase and H2O2 on the methoxylated ring of DMHB yields a cation radical, which then cleaves to give either benzaldehyde and an alpha-hydroxy(dimethoxybenzyl) radical or veratraldehyde and an alpha-hydroxybenzyl radical (cf. Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612; Snook, M. E., and Hamilton, G. A. (1974) J. Am. Chem. Soc. 96, 860-869). Similar mechanisms probably apply to the enzymatic C alpha-C beta cleavage of natural lignin.

Basidiomycota↗
PubMed1985

Oxidation of persistent environmental pollutants by a white rot fungus.

The white rot fungus Phanerochaete chrysosporium degraded DDT [1,1,-bis(4-chlorophenyl)-2,2,2-trichloroethane], 3,4,3',4'-tetrachlorobiphenyl, 2,4,5,2',-4',5'-hexachlorobiphenyl, 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocylohexane), and benzo[a]pyrene to carbon dioxide. Model studies, based on the use of DDT, suggest that the ability of Phanerochaete chrysosporium to metabolize these compounds is dependent on the extracellular lignin-degrading enzyme system of this fungus.

Carbon Dioxide↗
PubMed1985

The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes.

The hemoprotein ligninase of Phanerochaete chrysosporium catalyzes, in the presence of H2O2, a variety of seemingly different oxidations in lignin and lignin model compounds. Here we show that the enzyme also catalyzes the oxidation of various methoxybenzenes. ESR spectroscopy shows that the compounds are oxidized to aryl cation radicals. These decompose, evidently by H2O addition. Thus, 1,4-dimethoxybenzene is converted to p-benzoquinone and methanol. We propose a unified mechanism, based on formation of aryl cation radicals, to explain the various reactions catalyzed by the ligninase.

Anisoles↗
PubMed1985

Free hydroxyl radical is not involved in an important reaction of lignin degradation by Phanerochaete chrysosporium Burds.

Hydroxyl radical (HO.) has been implicated in the degradation of lignin by Phanerochaete chrysosporium. This study assessed the possible involvement of HO. in degradation of lignin substructural models by intact cultures and by an extracellular ligninase isolated from the cultures. Two non-phenolic lignin model compounds [aryl-C(alpha)HOH-C(beta)HR-C(gamma)H2OH, in which R = aryl (beta-1) or R = O-aryl (beta-O-4)] were degraded by cultures, by the purified ligninase, and by Fenton's reagent (H2O2 + Fe2+), which generates HO.. The ligninase and the cultures formed similar products, derived via an initial cleavage between C(alpha) and C(beta) (known to be an important biodegradative reaction), indicating that the ligninase is responsible for model degradation in cultures. Products from the Fenton degradation were mainly polar phenolics that exhibited little similarity to those from the biological systems. Mass-spectral analysis, however, revealed traces of the same products in the Fenton reaction as seen in the biological reactions; even so, an 18O2-incorporation study showed that the mechanism of formation differed. E.s.r. spectroscopy with a spin-trapping agent readily detected HO. in the Fenton system, but indicated that no HO. is formed during ligninase catalysis. We conclude, therefore that HO. is not involved in fungal C(alpha)-C(beta) cleavage in the beta-1 and beta-O-4 models and, by extension, in the same reaction in lignin.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium.

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.

Journal Article↗
PubMed1984

Lignin-degrading enzyme from Phanerochaete chrysosporium: Purification, characterization, and catalytic properties of a unique H(2)O(2)-requiring oxygenase.

An extracellular lignin-degrading enzyme from the basidiomycete Phanerochaete chrysosporium Burdsall was purified to homogeneity by ion-exchange chromatography. The 42,000-dalton ligninase contains one protoheme IX per molecule. It catalyzes, nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds: C(alpha)-C(beta) cleavage in lignin-related compounds of the type aryl-C(alpha)HOH-C(beta)HR-C(gamma)H(2)OH (R = -aryl or -O-aryl), oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in phenylglycol structures, and hydroxylation of benzylic methylene groups. It also catalyzes oxidative coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. All reactions require H(2)O(2). The C(alpha)-C(beta) cleavage and methylene hydroxylation reactions involve substrate oxygenation; the oxygen atom is from O(2) and not H(2)O(2). Thus the enzyme is an oxygenase, unique in its requirement for H(2)O(2).

Journal Article↗
PubMed1983

The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron.

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that O2-., H2O2, or the .OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.

Adenosine Monophosphate↗
PubMed1983

Effect of hydrogen peroxide on the initiation of microsomal lipid peroxidation.

Hydrogen peroxide reacts with reduced transition metals to generate the highly reactive hydroxyl radical (X OH), most often proposed as the predominant species for initiating microsomal lipid peroxidation. To assess the potential involvement of X OH, generated from hydrogen peroxide, in microsomal lipid peroxidation, we have altered the concentration of microsomal hydrogen peroxide and measured the resulting rates of malondialdehyde production. Hydrogen peroxide concentration in microsomes was changed by adding exogenous catalase, by washing to reduce both endogenous catalase activity and hydrogen peroxide-dependent glutathione oxidase activity, and by inhibiting endogenous catalase activity with azide in either the presence or absence of exogenous hydrogen peroxide. In only one instance was the rate of lipid peroxidation affected; exogenous hydrogen peroxide added to microsomes, previously incubated with azide, inhibited lipid peroxidation, the opposite effect from that predicted if X OH, generated from hydrogen peroxide, is actually the major initiating species. Neither these results, nor the inability of known X OH traps to inhibit microsomal lipid peroxidation, support the role of free hydrogen peroxide in the initiation of microsomal lipid peroxidation.

Animals↗
PubMed1982

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium.

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.

Agaricales↗
PubMed1982

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation.

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.

Adenosine Diphosphate↗