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.     

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.     

固定化胞外邻苯二酚1，2－双加氧酶的研究

首次将胞外邻苯二酚1,2－双加氧酶固定化,并用于制备顺,顺—己二烯二酸.该固定化酶表观活力高,使用范围扩大,耐酸性及耐碱性都有显著提高,并且使用稳定性好,得到的产物浓度及纯度均较高,酶与产物容易分离,整个工艺简单、独特、新颖,有利于工业化应用.     

Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.     

Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol.

Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

Determination of chrysene degradation under saline conditions by Fusarium sp. F092, a fungus screened from nature

Sixty-two rotted wood and soil samples were used to screen for chrysene-degrading fungi. A strain of Fusarium, named F092, was identified as most capable of degrading chrysene. F092 was active under saline and nonsaline conditions, breaking down 48% of the chrysene in 30 d. The percentage of chrysene degraded did not change at 35‰ salinity with pH 8.2 in solid and liquid cultures. The degradation under saline conditions increased about 0.6- and 2.1-fold in cultures with polypeptone and Tween80, and 0.03-fold in agitated cultures. F092 secreted nonligninolytic enzymes named 1,2-dioxygenase and 2,3-dioxygenase. The level of 1,2-dioxygenase activity reached 203.5 U L(-1) at 30 d and that of 2,3-dioxygenase activity, 29.7 U L(-1) at 40 d. The degradation pathway was clarified from the intermediates produced; chrysene 1,2-oxide, chrysene trans-1,2-dihydrodiol, 1-hydroxy 2-naphtoic acid, and catechol. F092 is a potential degrader of chrysene for bioremediation.     

A catabolic pathway for the degradation of chrysene by Pseudoxanthomonas sp. PNK-04

The chrysene-degrading bacterium Pseudoxanthomonas sp. PNK-04 was isolated from a coal sample. Three novel metabolites, hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid and salicylic acid, were identified by TLC, HPLC and MS. Key enzyme activities, namely 1-hydroxy-2-naphthoate hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase, were noted in the cell-free extract. These results suggest that chrysene is catabolized via hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid, salicylic acid and catechol. The terminal aromatic metabolite, catechol, is then catabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed catabolic pathway for chrysene degradation by strain PNK-04 is chrysene → hydroxyphenanthroic acid → 1-hydroxy-2-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol →cis,cis-muconic acid.     

Degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS

A bacterium was isolated from water by enrichment on 2-chlorobenzoate as sole source of carbon and energy. Based on morphological and physiological properties, this microorganism was assigned to the species Pseudomonas cepacia. The organism was designated Pseudomonas cepacia 2CBS. During growth on 2-chlorobenzoate, the chlorine substituent was released quantitatively, and a small amount of 2,3-dihydroxybenzoate accumulated in the culture medium. Mutants of Pseudomonas cepacia 2CBS were induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Some of these mutants produced catechol from 2-chlorobenzoate. Other mutants accumulated the meta-cleavage product of catechol, 2-hydroxy-cis,cis-muconic acid semialdehyde. In crude cell-free extracts of Pseudomonas cepacia 2CBS, an enzyme was detected which catalysed the conversion of 2-chlorobenzoate to catechol. Molecular oxygen, NADH and exogenous Fe2+ were required for activity. Stoichiometric amounts of chloride were released. Experiments with 18O2 revealed that both oxygen atoms in the hydroxyl groups of the product were derived from molecular oxygen. Thus, the enzyme catalysing the conversion of 2-chlorobenzoate was identified as 2-chlorobenzoate 1,2-dioxygenase (1,2-hydroxylating, dehalogenating, decarboxylating). 2-Chlorobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS was shown to be a multicomponent enzyme system. The activities of catechol 2,3-dioxygenase and catechol 1,2-dioxygenase were detected in crude cell-free extracts. The activity of catechol 2,3-dioxygenase was 60 times higher than the activity of catechol 1,2-dioxygenase, indicating that catechol is mainly degraded via meta-cleavage in Pseudomonas cepacia 2CBS. No enzyme was found which converted 2,3-dihydroxybenzoate, suggesting that this compound is a dead-end metabolite of 2-chlorobenzoate catabolism. A pathway for the degradation of 2-chlorobenzoate by Pseudomonas cepacia 2CBS is proposed.     

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.