Biochemical and genetic evidence for meta-ring cleavage of 2,4,5-trihydroxytoluene in Burkholderia sp strain DNT

2,4,5-Trihydroxytoluene (THT) oxygenase from Burkholderia sp, strain DNT ca talyzes the conversion of THT to an unstable ring fission product. Biochemi cal and genetic studies of THT oxygenase were undertaken to elucidate the m echanism of the ring fission reaction, The THT oxygenase gene (dntD) was pr eviously localized to the 1.2-kb DNA insert subcloned in the recombinant pl asmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831- 1837, 1993), Analysis of the deduced amino acid sequence of DntD revealed t he presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD cor responded to a molecular mass of 35 kDa, The native molecular masses for th e THT oxygenase estimated by using gel filtration chromatography and nonden aturing gel electrophoresis were 67.4 and 77.8 kDa, respectively, The resul ts suggested that the native protein consists of two identical subunits, Th e colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requ irement for ferrous iron in the active site. The properties of the enzyme a re similar to those of the catechol 2,3-dioxygenases (meta-cleavage dioxyge nases). In addition to THT, the enzyme exhibited activity towards 1,2,4-ben zenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol, The chemical analysis of the THT ring cleavage product showed that the prod uct was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.