Protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus

Hou, Ching T.; Lillard, Marjorie O.; Schwartz, Robert D.

doi:10.1021/bi00648a020

Cited by 54 publications

(30 citation statements)

References 24 publications

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“…This compares with a final specific activity of 105 U/mg for the A. radiobacter 3,4-PCD (23). Lower specific activities (ranging from 7 to 47 U/mg) have been observed for most purified 3,4-PCDs from other bacteria (4,6,14,15,18,23,29,33,59,64). The final purified streptomycete 3,4-PCD on a native PAGE gel was observed as a single protein band (data not shown), and on a corresponding SDS-PAGE gel, two proteins with approximate masses of 24.2 and 33.7 kDa were observed (Fig.…”

Section: Resultsmentioning

confidence: 75%

“…3). This falls in the lower end of the molecular size range of other 3,4-PCDs, 150 to 700 kDa (4,6,14,15,18,23,29,33,59,64). Based on an ␣-subunit size of 21.8 kDa and a ␤-subunit size of 29.3 kDa, as predicted by the gene products below, and what is known of characterized 3,4-PCDs, the native streptomycete enzyme is proposed to contain three ␣␤ protomers.…”

Section: Resultsmentioning

confidence: 89%

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Characterization of the Protocatechuic Acid Catabolic Gene Cluster from Streptomyces sp. Strain 2065

Iwagami

Yang

Davies

2000

Appl Environ Microbiol

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Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the ␤-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the ␤-and ␣-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6.5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused ␥-carboxymuconolactone decarboxylase/␤-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G؉C gram-positive bacteria and eukaryotes.The intradiol or ortho ring cleavage pathway commonly known as the ␤-ketoadipate pathway, named after the intermediate ␤-ketoadipate (3-oxoadipate) (57), is widely distributed among taxonomically diverse soil microorganisms, including both eubacteria and fungi. It is considered a "major utility pathway" playing a significant role in the processing and degradation of aromatic compounds from plant material found in soil, such as those originating from the solubilization of lignin (27). The pathway consists of two branches, one starting at catechol and the other at protocatechuic acid, which are cleaved by catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase (3,4-PCD), respectively. In bacteria the two branches of the pathway converge at the intermediate ␤-ketoadipate enol-lactone. The ␤-ketoadipate pathway is biochemically conserved, and the structural genes encoding enzymes of this pathway ( Fig. 1) in widely differing bacterial species are similar. The genes of the ␤-ketoadipate pathway have been cloned and sequenced from different bacteria, including Acinetobacter sp. strain ADP1 and Pseudomonas putida, two organisms whose GϩC contents differ by 20%, but amino acid sequence identities for isofunctional Pca enzymes range from 45 to 68% (27). Despite this conservation, diversity in the ␤-ketoadipate pathways has evolved in pathway branching, inducing metabolites, genetic organization, operon clustering, and regulation (48).In addition to its role in the degradation of aromatic compounds derived from lignin and other plant compounds that are recalcitrant or resistant to degradation, the enzymes of the ␤-ketoadipate pathway are required for the degradation of chlorocatechols by the modified ortho pathway. Although a 3,4-dihydroxychlorobenzoic acid ortho-cleaving enzyme has not been found, two protocatechuate 3,4-dioxygenase isozymes that oxidize 4-sulfocatechol were identified recently from two members ...

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Section: Resultsmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 89%

Characterization of the Protocatechuic Acid Catabolic Gene Cluster from Streptomyces sp. Strain 2065

Iwagami

Yang

Davies

2000

Appl Environ Microbiol

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“…1). A similar reaction is catalyzed by protocatechuate 3,4-dioxygenase (EC 1.13.11.3) (9,12,39,40), product of the chromosomal pcaA gene (8,11,15) (Fig. 1).…”

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confidence: 83%

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions

et al. 1988

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The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.

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“…3). The material eluting in the protocatechuate oxygenase (product of the pcaA gene) peak was judged to be nearly homogeneous by its behavior on different HPLC columns, but its specific activity of 4 jxmol/min per mg of protein was low compared with the specific activity of 20 ,umol/min per mg of protein found with homogeneous protocatechuate oxygenase purified from A. calcoaceticus (11) quantitatively converted 100 M protocatechuate toketoadipate in about 200 min, a rate corresponding to about three times the rate that would be predicted for the transformation at room temperature. The relatively high rate probably is due to the fact that the enzymatic conversion by whole cells was conducted at 370C.…”

Section: Resultsmentioning

confidence: 99%

Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus

Doten¹,

Ngai²,

Mitchell³

et al. 1987

J Bacteriol

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The lB-ketoadipate pathway of Acinetobacter caloaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. caloaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, ,B-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pea genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pea gene products at levels 10-to 30-fold higher than found in fully induced A. calcoaceicus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pea genes quantitatively converted protocatechuate to (-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for 0-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the ll-kbp fragment and derivatives of the ll-kbp fragment containing TnS in the pcaA, pcaB, peaC, pcaD, and peaE genes. The fragment containing the pea gene hybridized strongly and specifically to a previously cloned fragment containing A. caloaceticus cat genes.The pca structural genes encode six enzymes that convert protocatechuate to citric acid cycle intermediates via Pketoadipate ( Fig. 1; 27). Protocatechuate induces coordinate synthesis of the enzymes in Acinetobacter calcoaceticus (3), and their unified transcriptional control has been suggested by their constitutive formation in regulatory mutants (4,22). The physical organization of the A. calcoaceticus pca genes has not been explored. The A. calcoaceticus cat genes encode six enzymes that convert catechol to citric acid cycle intermediates by reactions analogous or identical to those catalyzed by the pca gene products (Fig. 1) To establish a basis for analysis of possible interaction between cat and pca genes in A. calcoaceticus, we constructed expression libraries of A. calcoaceticus DNA and used immunological screening to identify a clone that expressed the pcaE gene. The cloned DNA was an 11-kbp EcoRI fragment that hybridized with the 5-kbp EcoRI fragment containing the catBCDE genes and pos...

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Protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus

Cited by 54 publications

References 24 publications

Characterization of the Protocatechuic Acid Catabolic Gene Cluster from Streptomyces sp. Strain 2065

Characterization of the Protocatechuic Acid Catabolic Gene Cluster from Streptomyces sp. Strain 2065

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions

Cloning and genetic organization of the pca gene cluster from Acinetobacter calcoaceticus

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