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

Abstract

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.

FIG. 1

Schematic representation of the genomic region of strain TFA involved in tetralin biodegradation and the sequenced gene. Arrows represent divergent operons. Triangles represent locations of KIXX insertions in mutants unable to grow on tetralin as the only carbon source.

FIG. 2

Dendrogram showing the best tree obtained by the neighbor-joining method from the alignment of 26 sequences showing high similarity to ThnC. XylE MT2 was used as an outgroup. The scale represents distance expressed as percent divergence. The numbers at nodes are bootstrap values expressed as percentages. The ThnC sequence is boxed. The other sequences and their GenBank accession numbers are as follows: BphC PS400, Burkholderia cepacia LB400 2,3-DHBDOX (X66122); BphC PS707, P. pseudoalcaligenes KF707 2,3-DHBDOX (M83673); BphC OU83, P. putida OU83 2,3-DHBDOX (X91876); BphC PS715, P. putida KF715 2,3-DHBDOX (M33813); BphC PS102, Pseudomonas sp. strain KKS702 2,3-DHBDOX (M26443); CumC IP101, P. fluorescens IP101 catechol 2,3-dioxygenase (D37828); BphC JR1, Pseudomonas sp. strain JR1 2,3-DHBDOX (U53507); TodE PSF1, P. putida F1 3-methylcatechol 2,3-dioxygenase (JO4996); BphC RGA1, Rhodococcus sp. strain RHA1 2,3-DHBDOX (D32142); BphC1 RGP6, Rhodococcus globerulus P6 2,3-DHBDOX I (X75633); BphC M5, Rhodococcus sp. strain M5 2,3-DHBDOX (U27591); PcbC DJ12, Pseudomonas sp. strain DJ12 2,3-DHBDOX (D44550); CarC CB3, Sphingomonas sp. strain CB3 extradiol dioxygenase involved in carbazole degradation (AF060489); DbfB SPRW1, S. paucimobilis RW1 2,2′,3-trihydroxybiphenyl dioxygenase (X72850); NahC PSG7, P. putida PpG7 1,2-DHNDOX (J04994); PahC OUS82, P. putida OUS82 polycyclic aromatic hydrocarbon extradiol dioxygenase (D16629); NahC pNPL41, P. putida 1,2-DHNDOX (Y14173); DoxG PSC18, Pseudomonas sp. strain C18 1,2-DHNDOX (M60405); NahC PSA10, P. stutzeri AN10 1,2-DHNDOX (AF039533); NagC pWWU2, Pseudomonas sp. strain U2 1,2-DHNDOX in plasmid pWWU2 (AF036940); BphC SPQ1, S. yanoikuyae Q1 2,3-DHBDOX (M20640); BphC SPB1, S. yanoikuyae B1 2,3-DHBDOX (U23374); PhnQ DJ77, Pseudomonas sp. DJ77 2,3-DHBDOX (AF061802); BphC pNL1, S. aromaticivorans F199 2,3-DHBDOX in plasmid pNL1 (AF079317); NahC SPBN6, Sphingomonas sp. strain BN6 1,2-DHNDOX (U65001 [This sequence corresponds to the enzyme purified as described in reference 25); EtbC RGA1, Rhodococcus sp. strain RHA1 alternative 2,3-DHBDOX (D78322); XylE MT2, P. putida mt-2 catechol 2,3-dioxygenase (V01161).

FIG. 3

Polyacrylamide gel showing overproduction and purification of native or His-tagged ThnC. The lanes contained boiled whole induced cells of NCM631/pIZ227 bearing pIZ578 (lane 1, vector), pIZ590 (lane 2, native ThnC), or pIZ591 (lane 3, His-tagged ThnC); crude extracts of NCM631/pIZ227 bearing pIZ591 after breakage and centrifugation (lane 4); and purified His-tagged ThnC after affinity chromatography (lane 5). For induction conditions, see Materials and Methods.

FIG. 4

(A) Variation of the extinction coefficients of 4-(2-oxocyclohexyl)-2-hydroxy-buta-2,4-dienoic acid at the two absorption maxima as a function of pH. (B) Mass spectrum of the product resulting from the incubation of 4-(2-oxocyclohexyl)-2-hydroxy-buta-2,4-dienoic acid with ammonium chloride.

FIG. 5

Reaction catalyzed by ThnC in the tetralin biodegradation pathway of S. macrogoltabidus TFA.

FIG. 6

(A) Inactivation of ThnC in elution buffer (■) or in elution buffer plus 0.1 mM MnCl₂ (●) after incubation on ice and reactivation of the enzyme by the addition of 2 mM ferrous ammonium sulfate (□, ○). Arrows show times of addition of Fe²⁺. (B) Lineweaver-Burk plot of ThnC activity using DHT as the substrate. The inset represents the plot of [S]/V versus [S] and computer fitting of the data to the polynomial expression from which kinetic parameters were calculated.