Molecular Cloning and Biochemical Characterization of Protocatechuate 3,4-dioxygenase in Burkholderia sp. NCIMB 10467

Abstract:

Strain NCIMB 10467, a lignin degrader, was reclassified as genus Burkholderia according to its 16S rDNA sequence. It seems that the metabolism of protocatechuate by this strain is diverse under the induction of various substrates. A 9505-bp DNA fragment extending from a conserved region of the gene, which encodes a subunit of ortho cleavage protocatechuate 3,4-dioxygenase (P34D; EC 1.13.11.3), was obtained by genome walking. Sequence analysis revealed two deduced open reading frames, pcaG and pcaH, encoding the a and b subunits of P34D respectively in this fragment. The P34D activity could be detected when pcaGH were expressed in E. coli and the disruption of pcaH in strain NCIMB 10467 has lead to loss of its ability to catabolize protocatechuate. It was proved that the cloned pcaGH were encoding a functional protocatechuate 3,4-dioxygenase which was necessary for the protocatechuate metabolism in this strain.