Anaerobic oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium.

Anoxic cell extracts of a denitrifying bacterial isolate (PC-07) were shown to oxidize p-cresol to p-hydroxybenzoate. Oxidation of the substrate was independent of molecular oxygen and required nitrate as the natural terminal electron acceptor. Two enzyme activities were implicated in the pathway utilized by PC-07. A p-cresol methylhydroxylase mediated the oxidation of p-cresol to p-hydroxybenzaldehyde, which was further oxidized to p-hydroxybenzoate by an NAD+-dependent dehydrogenase. The PC-07 methylhydroxylase was partially purified by anion-exchange chromatography. The protein appeared to be a multifunctional flavocytochrome, which first oxidized p-cresol to p-hydroxybenzyl alcohol, which was then oxidized to p-hydroxybenzaldehyde. The identity of the aldehyde was confirmed by mass spectroscopy. The PC-07 methylhydroxylase had a limited substrate range and required an alkyl-substituted phenolic ring with a hydroxyl group in the para position. From the available evidence, p-cresol, a naturally occurring phenol, exhibited the greatest affinity to the enzyme and therefore may be its natural substrate.     

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1.

Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2+, oxidized toluene to p-cresol as the first detectable product. Experiments with p-deutero-toluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 18O2, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.     

The purification and properties of p-cresol-(acceptor) oxidoreductase (hydroxylating), a flavocytochrome from Pseudomonas putida.

The enzyme that catalyses the hydroxylation of the methyl group of p-cresol was purified from Pseudomonas putida. It has mol.wt. 115000 and appears to contain two subunits of equal molecular weight. One subunit is a c-type cytochrome and the other is a flavoprotein. Reduction of the cytochrome occurred on addition of substrate. The same enzyme catalyses both p-cresol hydroxylation and the further oxidation of the product, 4-hydroxybenzyl alcohol. The stoicheiometry of acceptor reduced per molecule of substrate oxidized is that for two dehydrogenation reactions. The Km for p-cresol is 7.3 x 10(-6) M and that for 4-hydroxybenzyl alcohol is 47.6 x 10(-6) M. The enzyme, which is assayed with phenazine methosulphate as electron acceptor, was stimulated by particulate material, which probably contains the acceptor in vivo.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

Biodegradation of cyclic amines by a Pseudomonas strain involves an amine mono-oxygenase

Pseudomonas putida O1G3 catalyzes the degradation of pyrrolidine and piperidine. This strain can use these compounds as the sole source of carbon, nitrogen, and energy. When the cyclic amines were used as the growth substrates, the synthesis of a soluble heme amine mono-oxygenase was induced in this bacteria. This observation was confirmed by spectrophotometric analysis and specific inhibitor. This mono-oxygenase is a NADH-dependent enzyme and catalyzes the cleavage of the C-N bond of the pyrrolidine and piperidine ring by a mechanism similar to a N dealkylation. This reaction could be followed by ring cleavage to form gamma-aminobutyraldehyde oxidized to gamma-aminobutyrate. Further investigations to purify the heme-containing mono-oxygenase are in progress.     

Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.     

Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde.

Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.     

Dissimilation of aromatic compounds by Alcaligenes eutrophus

The range of aromatic compounds that support the growth of Alcaligenes eutrophus has been determined, and the pathways used for the dissimilation of these substrates have been explored, largely by enzymatic analyses. The beta-ketoadipate pathway operates in the dissimilation of benzoate and p-hydroxybenzoate; the genetisate pathway, in the dissimilation of m-hydroxybenzoate; and the meta cleavage pathway, in the dissimilation of phenol and p-cresol. l-Tryptophan is oxidized via anthranilate; but the metabolic fate of anthranilate was not established. The metabolism of the three stereoisomers of muconic acid was also examined.     

The metabolism of cresols by species of Pseudomonas

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.     

Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.     

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.     

Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism

The degradation of the toxic phenol p-cresol by Pseudomonas bacteria occurs by way of the protocatechuate metabolic pathway. The first enzyme in this pathway, p-cresol methylhydroxylase (PCMH), is a flavocytochrome c. The enzyme first catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol, utilizing one atom of oxygen derived from water, and yielding one molecule of reduced FAD. The reducing electron equivalents are then passed one at a time from the flavin cofactor to the heme cofactor by intramolecular electron transfer, and subsequently to cytochrome oxidase within the periplasmic membrane via one or more soluble electron carrier proteins. The product, p-hydroxybenzyl alcohol, can also be oxidized by PCMH to yield p-hydroxybenzaldehyde. The fully refined X-ray crystal structure of PCMH in the native state has been obtained at 2. 5 A resolution on the basis of the gene sequence. The structure of the enzyme-substrate complex has also been refined, at 2.75 A resolution, and reveals significant conformational changes in the active site upon substrate binding. The active site for substrate oxidation is deeply buried in the interior of the PCMH molecule. A route for substrate access to the site has been identified and is shown to be governed by a swinging-gate mechanism. Two possible proton transfer pathways, that may assist in activating the substrate for nucleophilic attack and in removal of protons generated during the reaction, have been revealed. Hydrogen bonding interactions between the flavoprotein and cytochrome subunits that stabilize the intramolecular complex and may contribute to the electron transfer process have been identified.     

Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pathway. The initial steps in the degradative pathway lead to formation of 2,4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substituted catechols found in pathways for aromatic compound degradation. To determine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying the cloned gene. The findings were compared to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT and to other previously described ring cleavage dioxygenases. The comparison revealed that only 60% identity was shared by the two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytoluene as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dihydroxybiphenyl. However, results from enzyme assays indicated a strong preference for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydroxytoluene oxygenase from Burkholderia sp. strain DNT, and the distant genetic relationship to other ring cleavage enzymes suggest that dntD evolved expressly to carry out trihydroxytoluene transformation.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸(ABA)生物合成一般有两条途径:C15直接途径和C40间接途径,前者经C15法呢焦磷酸(FPP)直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA,是高等植物ABA生物合成的主要途径.9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤,然后黄质醛被氧化形成一种酮,该过程需NAD为辅因子,酮再转变形成ABA-醛,ABA-醛氧化最终形成ABA.在该途径中,玉米黄质环氧化酶(ZEP)、9-顺式环氧类胡萝卜素双加氧酶(NCED)和醛氧化酶(AO)可能起重要作用.     

Functional identification of the gene locus (ncg12319 and characterization of catechol 1,2-dioxygenase in Corynebacterium glutamicum

Corynebacterium glutamicum assimilated phenol, benzoate, 4-hydroxybenzoate p-cresol and 3,4-dihydroxybenzoate. Ring cleavage was by catechol 1,2-dioxygenase when phenol or benzoate was used and by protocatechuate 3,4-dioxygenase when the others were used as substrate. The locus ncg12319 of its genome was cloned and expressed in Escherichia coli. Enzyme assays showed that ncg12319 encodes a catechol 1,2-dioxygenase. This catechol 1,2-dioxygenase was purified and accepted catechol, 3-, or 4-methylcatechols, but not chlorinated catechols, as substrates. The optimal temperature and pH for catechol cleavage catalyzed by the enzyme were 30 degrees C and 9, respectively, and the Km and Vmax were determined to be 4.24 micromol l(-1) and 3.7 micromol l(-1) min(-1) mg(-1) protein, respectively.     

Identification of the cleavage sites of oxidized protein that are susceptible to oxidized protein hydrolase (OPH) in the primary and tertiary structures of the protein

Amino acid sequences in H(2)O(2)-oxidized bovine serum albumin (BSA) that are susceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 22, 20, and 8 kDa) were produced as analyzed by SDS-PAGE. N-Terminal amino acid sequence analysis of these fragments indicated that oxidized BSA was cleaved by OPH at three major sites, Leu218-Ser219, Tyr410-Thr411, and Phe506-Thr507, at an early stage of the proteolytic degradation. In the three-dimensional structure of BSA deduced by computer modeling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxidation on the tertiary structure of BSA was then investigated by hypothetically replacing all the four methionine and two tryptophan residues with their oxidized forms, methionine sulfoxide and N'-formyl-kynurenine, respectively. The three-dimensional structure of the hypothetically oxidized BSA indicated that all the three cleavage sites in the protein could become more exposed to the solvent than in unoxidized BSA. These results suggest that, upon oxidation of BSA, the amino acid sequences that are potentially cleavable by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating the cleavage sites of oxidized protein by oxidized protein-selective protease, suggesting the possible mechanism of oxidized protein-selective degradation by the enzyme.     

p-Cymene pathway in Pseudomonas putida: ring cleavage of 2,3-dihydroxy-p-cumate and subsequent reactions.

It was confirmed that 2,3-dihydroxy-p-cumate is a substrate for ring cleavage in Pseudomonas putida PL-W after growth with p-cymene or p-cumate. This compound was oxidized to pyruvate, acetaldehyde, isobutyrate, and carbon dioxide by extracts of cells, and these products appear in equimolar amounts. The transient appearance of compounds and 2,3-dihydroxy-p-cumate to a yellow intermediate (lambda max, 345 nm) without decarboxylation. Extracts of the benzene nucleus; this is followed by decarboxylation to give the 393-nm species, which gives rise to isobutyrate, acetaldehyde, and pyruvate by the hydrolytic route of meta cleavage of catechols, via 4-hydroxy-2-oxovalerate. This was confirmed with a mutant of P. putida PL-RF-1 that was unable to grow with p-cymene (or p-cumate) but was able to oxidize both compounds AND 2,3-DIHYDROXY-P-CUMATE TO A YELLOW INTERMEDIATE (LAMBDA MAX, 345 NM) WITHOUT DECARBOXYLATION. Extrats of P. putida PL-W (wild type) or a revertant of the mutant PL-RF-1 catalyzed the decarboxlation of the 345-nm intermediate with transient formation of the compound that absorbed at 393 nm. The substrate specificities of the 3,4-dioxygenative ring cleavage enzyme, and the decarboxylase were determined in crude extracts of P. putida PL-W and Pseudomonas fluorescens 007. It was conclude that 3,4-dioxygenative cleavage and decarboxylation are sequential enzyme-catalyzed reactions common to both P. putida and P. fluorescens for the oxidation of 2,3-dihydroxybenzoates. Unlike P. putida PL-W, which exclusively use the hydrolase branch, P. fluorescens 007 uses the dehydrogenase branch of the meta pathways that diverge after ring cleavage and later converge at oxoenate intermediates.     

Biodegradation of cresol isomers in anoxic aquifers

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.     

Biodegradation of cresol isomers in anoxic aquifers.

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.