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 identified 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 define a subfamily of 1, 2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7, 8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (V(max), 40.5 U mg(-1); K(m), 18. 6 microM). The enzyme shows even higher activity with 1, 2-dihydroxynaphthalene and also significant activity toward 1, 2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1, 2-dihydroxynaphthalene dioxygenases.
Andújar
E
E
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Seville, Spain.
Hernáez
M J
MJ
Kaschabek
S R
SR
Reineke
W
W
Santero
E
E
D049308:Dioxygenases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009284:Naphthols / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011487:Protein Conformation; D017421:Sequence Analysis; D013379:Substrate Specificity; D013696:Temperature; D013764:Tetrahydronaphthalenes / Q000378:metabolism*
D009284:Naphthols; D013764:Tetrahydronaphthalenes; C050642:1,2-dihydroxynaphthalene; D010105:Oxygenases; C023133:dihydroxynaphthalene oxygenase; D049308:Dioxygenases; C095210:tetralin