Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox ) are related enzymes that have differentiated during evolution as their sp ecificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpo se of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox com ponents in the Nap dox system. Both enzyme systems were purified by affinit y chromatography as histidine-tagged fused proteins. Data show for the firs t time that Nap dox can catalyze the oxygenation of all three monochlorobip henyl isomers, but it is unable to hydroxylate 2,5-, 2,2'-, 3,3'-, 4,4'-di- and 2,2',5,5'-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph do x reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin . However, when the Bph dox ferredoxin was used to reconstitute a hybrid Na p dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases.