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Bacterial degradation of N-cyclopropylmelamine. The steps to ring cleavage.

ABSTRACT: The s-triazine cyclopropylmelamine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) was degraded to about 6 mol of NH4+/mol of substrate by a mixture of two bacteria (strains A and D, both Pseudomonas spp.) Only strain A grew with cyclopropylmelamine as sole and limiting source of nitrogen. The organism obtained 2 mol of nitrogen/mol of substrate and excreted a product that was identified as cyclopropylammelide [6-cyclopropylamino-1,3,5-triazine-2,4(1 H,3 H)-dione]. Proteins in extracts from strain A were separated on a Sephadex G-200 column. Cyclopropylmelamine was found to be deaminated in two separable steps to cyclopropylammelide via cyclopropylammeline [4-amino-6-cyclopropylamino-1,3,5-triazine-2(1 H)-one], which was identified. Strain D could not utilize cyclopropylmelamine or cyclopropylammeline, but could utilize cyclopropylammelide (or homologue) as sole and limiting source of nitrogen and obtain about 4 mol of nitrogen/mol of substrate. Proteins in cell extracts from strain D were separated on a DEAE-cellulose column. Alkylammelides were degraded quantitatively by one enzyme fraction to 1 mol of cyanuric acid plus 1 mol of alkylamine/mol of substrate. The specific activities of enzymes in extracts of the two strains were as high as the activities observed during growth. The three activities studied in the two strains were all active under aerobic and oxygen-free conditions. The reactions appear to be hydrolytic, yielding 2 mol of NH4+ plus 1 mol of cyclopropylamine and 1 mol of cyanuric acid/mol of substrate.

SUBMITTER: Cook AM

PROVIDER: S-EPMC1144181 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

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Project description:Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angular dioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the 2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1a-C-2 bond (decarboxylation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in the degradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of 1 molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygenated, resulting in the cleavage of the C-1a-C-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring cleavage of aromatic compounds.IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key and speed-limiting step in the aerobic degradation of aromatic compounds. As previously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihydroxylated at a lateral position and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis, the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), resulting in the cleavage of the C-1a-C-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe. Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of benzene ring cleavage.

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Bacterial degradation of N-cyclopropylmelamine. The steps to ring cleavage.

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