Identification of an extradiol dioxygenase involved in tetralin biodegradation: Gene sequence analysis and purification and characterization of the gene product

A genomic region involved in tetralin biodegradation was recently identifie d in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to def ine a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which c omprises two clearly different groups, and to show that ThnC clusters withi n group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene wa s found to be the metabolite accumulated by a thnC insertion mutant. The ri ng cleavage product of this metabolite exhibited behavior typical of a hydr oxymuconic semialdehyde toward pa-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purifie d, and its biochemical properties have been studied. The enzyme is a decame r which requires Fe(II) for activity and shows high activity toward its sub strate (V-max, 40.5 U mg(-1); K-m, 18.6 mu M). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity towar d 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specif icity of ThnC is consistent with that exhibited by other extradiol dioxygen ases of the same group within the subfamily of 1,2-dihydroxynaphthalene dio xygenases.