SUMMARY: The nucleotide sequence of the 3-chlorobenzoate 3,4-dioxygenase genes, designated ,from the transposon Tn5271 was determined. The function of the two sequenced open reading frames was evaluated by mutagenesis and expressionto show that theandgenes code for dioxygenase and reductase proteins, respectively. Comparison of the deduced amino acid sequences of the genes with sequences for other oxygenases revealed a clearly defined lineage among the class IA oxygenases that shows several unique features. This lineage includes phthalate 4,5-dioxygenase (), and based on the available NH-terminal sequence of component A, also includes 4-sulphobenzoate 3,4-dioxygenase. Vanillate demethylase, encoded by the genes and formally a monooxygenase enzyme catalysing an oxidative demethylation, is also included in this lineage. The terminal chlorobenzoate dioxygenase (CbaA) component is characterized by a conserved Rieske-type [2Fe-2S] ligand centre. The reductase component (CbaB) contains a plant-type ferredoxin [2Fe-2S] FMN-isoalloxazine and NAD-ribose-binding domains and the orientation of these domains is conserved in all known class IA reductases. These results support the hypothesis that alternative fusions of the electron transfer modules of the reductases arose early in the divergence of oxygenase systems. The over-riding evolutionary constraint acting on the divergence of the class IA oxygenases would appear to be the requirement for a carboxyl group to the site of oxygen insertion into the aromatic ring.


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