RESUMO
Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I). The latter gene was cloned and expressed in Escherichia coli. Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases. Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp. YL. Both enzymes share an unusual high temperature optimum of 65 degrees C. Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg-1 protein were obtained in E. coli, and putatively, owing to its own sigma70-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg-1 protein was measured.
Assuntos
Agrobacterium tumefaciens/enzimologia , Agrobacterium tumefaciens/genética , Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Expressão Gênica/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
We isolated and characterized D,L-halidohydrolases fromfive different soil bacteria. Three of these bacterial strains bear plasmidswith sizes of approximately 60 kb. Curing and mating experiments indicatedthat these three plasmids pFL160, pFL170, and pFL190 encoded a dehalogenase.Owing to their biochemical characterization, these halidohydrolases wereclosely related among each other and to the DhlIV halidohydrolase, encoded byplasmid pFL40 from Alcaligenes xylosoxidans ssp.denitrificans ABIV. Restriction enzyme patterns as well asDNA-hybridization experiments with an internal fragment of dhlIVrevealed a high degree of homology among each of these four plasmids andtheir dehalogenase genes.
RESUMO
Five bacterial strains were isolated from polluted soilscapable of degrading 2,2-dichloropropionate. In crude extracts, dehalogenaseactivity against haloacetates and longer-chained 2-haloalkanoic acids couldbe detected. Results from activity staining indicated that all bacterialstrains expressed a single dehalogenase. In further biochemicalcharacterization, two types of D,L-specific 2-haloalkanoic acid dehalogenaseswere described, which are different from each other not only in molecularweight and electrophoretic mobility, but also in sensitivity towards thiolreagents. Dehalogenases of these strains have been shown to be inducible andare catalyzing halide hydrolysis with inversion of product configuration.
RESUMO
We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L- isomer specific dehl dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a sigma 54-dependent activator protein, similar to the dehRI regulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.
Assuntos
Alcaligenes/genética , Genes Bacterianos , Hidrolases/genética , Alcaligenes/enzimologia , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Hidrolases/química , Hidrolases/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Pseudomonas fluorescens/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Estereoisomerismo , Reagentes de Sulfidrila/farmacologiaRESUMO
In the radiotherapy of malignant tumors of the oral cavity, the exact distribution of radiation dosage in the neighborhood of metallic dental replacement has been uncertain so far. Many authors therefore asked that crowns and bridges be removed before radiotherapy. By means of lithium fluoride thermo-luminescence dosimetry, punctate measurements of doses are possible for the first time. In view of the dose values obtained, removal of metallic dental replacement before the beginning of radiotherapy as a prophylactic measure to prevent radioosteomyelitis appears not to be necessary.