Analysis of Benzoate 1,2‐Dioxygenase Identifies Shared Electron Transfer Components With DxnA1A2 in <i>Rhizorhabdus wittichii</i> RW1

Rhizorhabdus wittichii RW1 is known for its ability to degrade polycyclic aromatic hydrocarbons, such as dibenzo-p-dioxin (DD) and dibenzofuran (DF). We hypothesized that the R. wittichii RW1 benzoate 1,2-dioxygenase shares electron transfer components with the DD/DF angular dioxygenase (DxnA1A2), similar to many aromatic hydrocarbon degrading sphingomonads. The genes encoding the benzoate oxygenase component (benAB) were identified in the RW1 genome sequence through homology to known benzoate oxygenases. The RW1 benAB genes are upstream from a putative benD gene encoding a cis-benzoate dihydrodiol dehydrogenase. Knockout of the benA gene resulted in a strain unable to grow on benzoate. The knockout strain could be complemented with the cloned benABD genes. Expression of benAB in Escherichia coli along with the fdx3 and redA2 genes, which encode the ferredoxin and reductase components utilized by DxnA1A2, produced a functional benzoate dioxygenase enzyme capable of converting benzoate to benzoate cis-dihydrodiol. Double knockout mutagenesis of the RW1 redA1 and redA2 reductase genes results in a mutant unable to grow on benzoate as the sole carbon source. Based on the gene knockout and heterologous expression experiments the RW1 benzoate 1,2 dioxygenase was identified and shares electron transfer components with DxnA1A2.