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Characterization and regulation of the genes for a novel anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1.

ABSTRACT: Anthranilate (2-aminobenzoate) is an important intermediate in tryptophan metabolism. In order to investigate the degradation of tryptophan through anthranilate by Burkholderia cepacia, several plasposon mutations were constructed of strain DBO1 and one mutant with the plasposon insertion in the anthranilate dioxygenase (AntDO) genes was chosen for further study. The gene sequence obtained from flanking DNA of the plasposon insertion site revealed unexpected information. B. cepacia DBO1 AntDO (designated AntDO-3C) is a three-component Rieske-type [2Fe-2S] dioxygenase composed of a reductase (AndAa), a ferredoxin (AndAb), and a two-subunit oxygenase (AndAcAd). This is in contrast to the two-component (an oxygenase and a reductase) AntDO enzyme from Acinetobacter sp. strain ADP1, P. aeruginosa PAO1, and P. putida P111. AntDO from strains ADP1, PAO1, and P111 are closely related to benzoate dioxygenase, while AntDO-3C is closely related to aromatic hydrocarbon dioxygenases from Novosphingobium aromaticivorans F199 and Sphingomonas yanoikuyae B1 and 2-chlorobenzoate dioxygenase from P. aeruginosa strains 142 and JB2. Escherichia coli cells expressing the functional AntDO-3C genes transform anthranilate and salicylate (but not 2-chlorobenzoate) to catechol. The enzyme includes a novel reductase whose absence results in less efficient transformation of anthranilate by the oxygenase and ferredoxin. AndR, a possible AraC/XylS-type transcriptional regulator, was shown to positively regulate expression of the andAcAdAbAa genes. Anthranilate was the only effector (of 12 aromatic compounds tested) that was able to induce expression of the genes.

SUBMITTER: Chang HK

PROVIDER: S-EPMC193950 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Characterization and regulation of the genes for a novel anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1.

Chang Hung-Kuang HK Mohseni Paria P Zylstra Gerben J GJ

Journal of bacteriology 20031001 19

Anthranilate (2-aminobenzoate) is an important intermediate in tryptophan metabolism. In order to investigate the degradation of tryptophan through anthranilate by Burkholderia cepacia, several plasposon mutations were constructed of strain DBO1 and one mutant with the plasposon insertion in the anthranilate dioxygenase (AntDO) genes was chosen for further study. The gene sequence obtained from flanking DNA of the plasposon insertion site revealed unexpected information. B. cepacia DBO1 AntDO (d ...[more]

PMID: 13129960

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Project description: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 (2-aminobenzoate) to catechol with insertion of both atoms of O(2) and consumption of one NADH. The terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. 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 degrees C) or nonpermissive (39 degrees 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 alpha and beta 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.

Dataset Information

Characterization and regulation of the genes for a novel anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1.

Publications

Characterization and regulation of the genes for a novel anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1.

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