The catechol 2,3-dioxygenase gene of Rhodococcus rhodochrous CTM: nucleotide sequence, comparison with isofunctional dioxygenases and evidence for an active-site histidine

Abstract

Summary: In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb BglII fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a catechol 2,3-dioxygenase (C230) accepting both 3-methylcatechol and 2,3-dihydroxy-biphenyl as substrates could be detected. The plasmid-encoded gene for C230 of R. rhodochrous CTM and its flanking regions were sequenced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The derived amino acid sequence of the C230 was compared to that of nine other enzymes, which all catalyse the extradiol cleavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Gram-positive bacterium. The role of four strongly conserved histidines was examined by chemical modification of the histidyl residues of the native enzyme by diethylpyrocarbonate. For that purpose the C230 was purified to homogeneity from E. coli harbouring pSC1701. However, the enzyme lost its activity during the purification. Activity could partially be restored by treatment with Fe²⁺and reducing agents.