EVOLUTIONARY RELATIONSHIP BETWEEN CHLOROCATECHOL CATABOLIC ENZYMES FROM RHODOCOCCUS-OPACUS 1CP AND THEIR COUNTERPARTS IN PROTEOBACTERIA - SEQUENCE DIVERGENCE AND FUNCTIONAL CONVERGENCE

Biochemical investigations of the muconate and chloromuconate cycloiso merases from the chlorophenol-utilizing strain Rhodococcus opacus (ery thropolis) 1CP had previously indicated that the chlorocatechol catabo lic pathway of this strain may have developed independently from the c orresponding pathways of proteobacteria. To test this hypothesis, we c loned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomer ase. Sequencing of the clones revealed that they comprise different pa rts of the same gene cluster in which five open reading frames hare be en identified, The clcB gene for chloromuconate cycloisomerase is tran scribed divergently from a gene which codes for a LysR-type regulatory protein, tile presumed ClcR, Downstream of clcR but separated from it by 222 bp, we detected the clcA and clcD genes, which could unambiguo usly be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hy drolase. A gene coding for a maleylacetate reductase could not be dete cted. Instead, the product encoded by ?he fifth open reading frame tur ned out to be homologous to transposition-related proteins of IS1031 a nd Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygen ases and cycloisomerases, respectively, clearly showed that the chloro catechol catabolic enzymes of R, opacus 1CP represent different branch es in the dendrograms than their proteobacterial counterparts, Thus wh ile the sequences diverged, the functional adaptation to efficient chl orocatechol metabolization occurred independently in proteobacteria an d gram-positive bacteria, that is, by functionally convergent evolutio n.