Characterization and Evolution of Anthranilate 1,2-Dioxygenase from Acinetobacter sp. Strain ADP1

Abstract

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O 2 consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O 2 consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23°C) or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the ␣ and ␤ subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

“…For example, Fetzner et al (17) obtained less than 0.5 mg of 2-HBADO per liter of culture of P. cepacia 2CBS. Although the oxygenase components of some ring-hydroxylating enzymes, including ANDO (14), have been overproduced in E. coli, the production of others appears to be limited by the formation of inclusion bodies (36,45), as was observed in expressing xylXY in E. coli. This may reflect the inability of E. coli strains to effectively incorporate the Fe 2 S 2 cluster into the dioxygenase components as they are translated.…”

“…Other class IB enzymes possessing different activities were subsequently discovered. For example, ANDO and 2-HBADO preferentially transform ortho-substituted benzoates, although the latter has a preference for benzoates with electron-withdrawing substituents and catalyzes the dehalogenation of ortho-chlorinated substrates (14,17). The availability of these related enzymes and the relative solubility of their substrates facilitate a range of studies to investigate structure-function relationships of ring-hydroxylating dioxygenases.…”

Aromatic degradative pathways in Acinetobacter baylyi underlie carbon catabolite repression

“…The antABC genes coding for anthranilate dioxygenase (Bundy et al, 1998;Eby et al, 2001) and the benABC genes coding for benzoate dioxygenase (Neidle et al, 1991) are homologous. The ben operon is regulated by BenM, a LysR family regulator able to activate transcription synergistically, responding to two effectors (benzoate and muconate) (Bundy et al, 2002;Collier et al, 1998;Ezezika et al, 2007a, b).…”

Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2

“…1). Based on the results of homology search of the database using the deduced amino acid sequences of the ORFs ORFI-1 ORFI-3, ORFI-4, and ORFI-5 are apparently the genes for the large subunit (benA) and the small subunit (benB) of the terminal oxygenase, the reductase component (benC) of the benzoate dioxygenase, and the DHB dehydrogenase (benD), respectively (8,12,14,20,25,26). The deduced amino acid sequences of ORFII-1, ORFII-3, and ORFII-4 are similar to catA, catB, and catC of the catechol degradation gene cluster of the proteobacteria, respectively ( Table 2), indicating that ORFII-1, ORFII-3, and ORFII-4 are the genes for catechol 1,2-dioxygenase (19, 23), CCM-lactonizing enzyme (41,42), and muconolactone isomerase (41,42), respectively.…”