Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

The catalytic cycle of catechol oxidase

Hybrid density functional theory with the B3LYP functional has been used to investigate the catalytic mechanism of catechol oxidase. Catechol oxidase belongs to a class of enzymes that has a copper dimer with histidine ligands at the active site. Another member of this class is tyrosinase, which has been studied by similar methods previously. An important advantage for the present study compared to the one for tyrosinase is that X-ray crystal structures exist for catechol oxidase. The most critical step in the mechanism for catechol oxidase is where the peroxide O–O bond is cleaved. In the suggested mechanism this cleavage occurs in concert with a proton transfer from the substrate. Shortly after the transition state is passed there is another proton transfer from the substrate, which completes the formation of a water molecule. An important feature of the mechanism, like the one for tyrosinase, is that no proton transfers to or from residues outside the metal complex are needed. The calculated energetics is in reasonable agreement with experiments. Comparisons are made to other similar enzymes studied previously.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.     

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ZD 4-3

Catechol 2,3-dioxygenase (C23O), an extradiol-type dioxygenase cleaving the aromatic C—C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxy-muconic semialdehyde. Based on a curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from Pseudomonas aeruginosa ZD 4-3, which was able to degrade both single and bicyclic compounds via a meta-cleavage path-way. A complete nucleotide sequence analysis of the C23O revealed that it has one ORF, which showed a strong overall amino acid similarity to the known gram-negative bacterial mesophilic C23Os. The alignment analysis indicated a distinct difference between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases that cleave bicyclic aromatic compounds. The heterogeneous expression of the pheB gene in E. Coli BL21(DE3) demonstrated that this C23O possesses a meta-cleavage activity.From Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 802–809.Original English Text Copyright © 2004 by Chen, Liu, Zhu, Jin.This article was submitted by the authors in English.     

PrP overdrive

Knockout of the cellular prion protein (PrP^C) in mice is tolerated, as is complete elimination of the protein’s N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP^C undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP^C fully encompasses the three α-cleavage sites. Analysis of all reported PrP^C deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein’s N-terminal residues, segments 23–31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP^C’s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP^C and consequent dyshomeostasis.     

Dienelactone hydrolase from Rhodococcus erythropolis 1 CP: purification and properties

Dienelactone hydrolases (EC 3.1.1.45) have been shown to play an indispensable role in the degradation of chloroaromatic compounds via ortho-cleavage of chlorocatechols. We report on the purification of dienelactone hydrolase of the chlorophenol-utilizing strain Rhodococcus erythropolis 1CP to apparent homogeneity. Dienelactone hydrolase differed fron the corresponding enzymes of other chloroaromatic compound-catabolizing strains in being restricted to substrates with a cis-dienelactone structure. From the cis-dienelactone-hydrolyzing enzyme of a 4-fluorobenzoate-utilizing Burkholderia (Pseudomonas) cepacia strain, it differed considerably in properties such as pH optimum of activity, inhibition by p-chloromercuribenzoate, and amino acid composition. Thus, there is not necessarily a close relationship between substrate specificity and other properties of dienelactone hydrolases.