Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli

Kälin, Markus; Neujahr, Halina Y.; Weissmahr, R N; Sejlitz, Torsten; Johl, Regula; Fiechter, Armin; Reiser, Jakob

doi:10.1128/jb.174.22.7112-7120.1992

Cited by 52 publications

(42 citation statements)

References 69 publications

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“…Putative ADP and FAD binding sequences of DntB aligned with those of other proteins (DntB, MNC monooxygenase from Burkholderia sp. strain DNT; TbuD [18], phenol-cresol hydroxylase from Pseudomonas pickettii PKO1; NahG [48], salicylate hydroxylase from P. putida PpG7; TfdB [29], 2,4-dichlorophenol hydroxylase from Alcaligenes eutrophus JMP134; PhyA [17], phenol hydroxylase from T. cutaneum; PobA [9], p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa; AlkT [8], rubredoxin reductase from Pseudomonas oleovorans; PheA [27], phenol 2-monooxygenase from Pseudomonas sp. strain EST1001; TodA [50], ferredoxin oxidoreductase of toluene dioxygenase from P. putida F1; BphG [10], ferredoxin oxidoreductase of biphenyl dioxygenase from Pseudomonas strain LB400; PcpB [28], pentachlorophenol-4-monooxygenase from Flavobacterium sp.…”

Section: Discussionmentioning

confidence: 99%

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Haigler¹,

Suen²,

Spain³

1996

J Bacteriol

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4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene byBurkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.Microorganisms can use either monooxygenase (33,34,36,49) or dioxygenase (2,15,21,25,33,37,40) enzymes to catalyze the oxidative removal of nitro groups from nitroaromatic compounds. Although the recent literature contains numerous examples of oxygenase-catalyzed nitro group displacements (33), little is known about the enzymes involved in these reactions. Monooxygenases that oxidize 4-and 2-nitrophenol (34, 36, 49) have been described, but only the 2-nitrophenol monooxygenase from Pseudomonas putida B2 has been purified and characterized (49). Recent evidence has suggested that some of the dioxygenases involved in the removal of nitro groups from aromatic compounds are multicomponent enzyme systems similar to the naphthalene dioxygenase enzyme system (2, 40). Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT) uses both mono-and dioxygenase enzymes to remove nitro groups (13,37,40) in an oxidative pathway that leads to the mineralization of 2,4-dinitrotoluene (2,4-DNT) (37).The initial attack on 2,4-DNT by...

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Section: Discussionmentioning

confidence: 99%

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Haigler¹,

Suen²,

Spain³

1996

J Bacteriol

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“…The gene (KRH_22060) adjacent to the probable meta-cleavage gene cluster may encode phenol 2-monooxygenase (EC 1.14.13.7) participating in the oxidation of phenol derivatives into o-diols, which are then channeled into the homoprotocatechuate catabolic pathway. The enzyme from Trichosporon cutaneum, a soil-living yeast, is known to possess a broad substrate specificity hydroxylating simple hydroxyl-, amino-, methyl-, and halogen-substituted phenols (17). The K. rhizophila DC2201 enzyme is similar (35% identity) to the eukaryotic enzyme from T. cutaneum, although the exact substrate specificity of the K. rhizophila enzyme is not known.…”

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confidence: 99%

Complete Genome Sequence of the Soil Actinomycete Kocuria rhizophila

et al. 2008

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The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae, a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G؉C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transfer, reflecting the small genome size. The presence of probable metabolic pathways for the transformation of phenolic compounds generated from the decomposition of plant materials, and the presence of a large number of genes associated with membrane transport, particularly amino acid transporters and drug efflux pumps, may contribute to the organism's utilization of root exudates, as well as the tolerance to various organic compounds.

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“…Quantification of the various bands seen in the Southern blot revealed that, if the DNA shown in Fig. 1B, (21) (35,66) and in genes from Neurospora crassa and Aspergillus species (2,80). Codons ending in A are avoided, and the ones ending in pyrimidines, especially C, are preferred.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations

et al. 1994

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Tnichosporon cutaneum belongs to genus Trichosporon Behrend, which was first described by Behrend in 1890 (3) and which is composed of a heterogeneous group of basidiomycetous yeasts containing organisms that differ from each other in a number of morphological, physiological, and genetic characteristics (25,27,28,45 (83) has been applied to a number of T. beigelii strains (39). The positive test results which were obtained are in agreement with the described basidiomycetous affinity of these strains. Tnichosporon yeasts have been isolated from a number of sources including soil, industrial wastewater, wood pulp, sludge, and clinical specimens, and T. beigelii has been found to be the causative agent of white piedra, which is a relatively inconse-* Corresponding author.

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Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli

Cited by 52 publications

References 69 publications

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Complete Genome Sequence of the Soil Actinomycete Kocuria rhizophila

Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations

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