Properties of the trihydroxytoluene oxygenase from Burkholderia cepacia R34: an extradiol dioxygenase from the 2,4-dinitrotoluene pathway

Burkholderia cepacia R34 mineralizes 2,4-dinitrotoluene via an oxidative pa thway. The initial steps in the degradative pathway lead to formation of 2, 4,5-trihydroxytoluene, which serves as the substrate for the ring cleavage dioxygenase. The trihydroxylated substrate differs from the usual substitut ed catechols found in pathways for aromatic compound degradation. To determ ine whether the characteristics of the trihydroxytoluene oxygenase reflect the unusual ring cleavage substrate of the 2,4-dinitrotoluene pathway, the gene encoding trihydroxytoluene oxygenase (dntD) was cloned and sequenced, and ring cleavage activity determined from recombinant bacteria carrying th e cloned gene. The findings were compared to the trihydroxytoluene oxygenas e from Burkholderia sp. strain DNT and to other previously described ring c leavage dioxygenases. The comparison revealed that only 60% identity was sh ared by the: two trihydroxytoluene oxygenases, but the amino acid residues involved in cofactor binding, catalysis, and protein folding were conserved in the DntD sequence. The enzyme catalyzed meta-fission of trihydroxytolue ne as well as the substrate analogues 1,2,4-benzenetriol, catechol, 3-methy lcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and 2,3-dih ydroxybiphenyl. However, results from enzyme assays indicated a strong pref erence for trihydroxytoluene, implying that it was the native substrate for the enzyme. The apparent enzyme specificity, its similarity to the trihydr oxytoluene oxygenase from Burkholderia in so. strain DNT, and the distant g enetic relationship to other ring cleavage enzymes suggest that dntD evolve d expressly to carry out trihydroxytoluene transformation.