THE CATECHOL 2,3-DIOXYGENASE GENE OF RHODOCOCCUS-RHODOCHROUS CTM - NUCLEOTIDE-SEQUENCE, COMPARISON WITH ISOFUNCTIONAL DIOXYGENASES AND EVIDENCE FOR AN ACTIVE-SITE HISTIDINE

In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb Bg/II fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a cat echol 2,3-dioxygenase (C230) accepting both 3-methylcatechol and 2,3-d ihydroxybiphenyl as substrates could be detected. The plasmid-encoded gene for C230 of R. rhodochrous CTM and its flanking regions were sequ enced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gen e in E. coli TG1. The derived amino acid sequence of the C230 was comp ared to that of nine other enzymes, which all catalyse the extradiol c leavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Cram-positive bacterium. The role of four strongly conserved histidi nes was examined tay 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 p artially be restored by treatment with Fe2+ and reducing agents.