Publications

• Purification and characterization of chlorobenzene cis-dihydrodiol dehydrogenase from Xanthobacter flavus 14p1.

  Spiess E., Gorisch H.

  Chlorobenzene cis-dihydrodiol dehydrogenase was purified to homogeneity from Xanthobacter flavus 14p1, which used 1,4-dichlorobenzene as the sole source of carbon and energy. The enzyme converted a number of halogenated substrates with high specific activity. The pI of the native chlorobenzene cis ... >> More

  Chlorobenzene cis-dihydrodiol dehydrogenase was purified to homogeneity from Xanthobacter flavus 14p1, which used 1,4-dichlorobenzene as the sole source of carbon and energy. The enzyme converted a number of halogenated substrates with high specific activity. The pI of the native chlorobenzene cis-dihydrodiol dehydrogenase was 5.4, and the molecular mass was approximately 100 kDa, as determined by gel filtration. The enzyme was composed of four apparently identical subunits with a molecular mass of 26.5 kDa. The Michaelis constant for 3,6-dichlorobenzene cis-dihydrodiol (210 microM) was lower than for benzene cis-dihydrodiol (780 microM), while the specific activity with benzene cis-dihydrodiol (63 units/ mg) was higher than with 3,6-dichlorobenzene cis-dihydrodiol (32 units/mg). Chlorobenzene cis-dihydrodiol dehydrogenase accepted also NADP+ as cosubstrate; however, the activity was reduced to 14% of that with NAD+. The enzymic activity was inhibited by mercuric chloride and to a lesser extent by the metal-ion chelators 8-hydroxyquinoline and KCN. << Less

  Arch Microbiol 165:201-205(1996) [PubMed] [EuropePMC]

• Transformation of chlorinated benzenes and toluenes by Ralstonia sp. strain PS12 tecA (tetrachlorobenzene dioxygenase) and tecB (chlorobenzene dihydrodiol dehydrogenase) gene products.

  Pollmann K., Beil S., Pieper D.H.

  The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformati ... >> More

  The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12. << Less

  Appl. Environ. Microbiol. 67:4057-4063(2001) [PubMed] [EuropePMC]

• Chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene.

  Pollmann K., Wray V., Pieper D.H.

  To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatecho ... >> More

  To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone. << Less

  J. Bacteriol. 187:2332-2340(2005) [PubMed] [EuropePMC]