SUBSTITUTION OF THE ISP ALPHA-SUBUNIT OF BIPHENYL DIOXYGENASE FROM PSEUDOMONAS RESULTS IN A MODIFICATION OF THE ENZYME-ACTIVITY

We have constructed a hybrid multicomponent dioxygenase gene cluster i n which the bphAl gene, coding for ISP alpha subunit of biphenyl dioxy genase from Pseudomonas pseudoalcaligenes KF707, has been replaced by the bedCl gene encoding the corresponding subunit of benzene dioxygena se from P. putida ML2. Escherichia coli cells containing the chimeric dioxygenase acquired the novel capability of producing indigo from ind ole. Furthermore, when compared to biphenyl dioxygenase, the hybrid di oxygenase enzyme was half as active towards benzene but exhibited only 4% activity when biphenyl was the substrate. The results implicate th e ISP alpha subunit to be involved in substrate specificity and activi ty of the dioxygenase enzyme. (C) 1994 Academic Press, Inc.