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

Abstract

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 ...

Genomic Analysis and Identification of Catabolic Pathways for Aromatic Compounds in Corynebacterium glutamicum

“…In terms of sequence identity, the genes involved in the protocatechuate branch of the -ketoadipate pathway are generally more similar to their counterparts in Gram-positive bacteria such as Streptomyces sp. and Rhodococcus opacus 13,21) . But significant differences in gene structure and organization were also found between C. glutamicum and Streptomyces sp.…”

Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

“…The accession numbers and references for the sequences from B. japonicum, Rhodococcus opacus 1CP, P. putida DOT-T1E, P. aeruginosa, Streptomyces sp. 2065 and S. coelicolor are O31385 (Lorite et al, 1998), AAC38245 (Eulberg et al, 1998), AAD39559 (Ramos et al, 1998), NP_248921 (Stover et al, 2000), AAD40814 (Iwagami et al, 2000) and T35016, respectively.…”

Degradation of naphthalene by thermophilic bacteria via a pathway, through protocatechuic acid

“…Protocatechuate 3, 4-dioxygenase (3, 4-PCDase; EC 1.13.11.3) activity was determined by monitoring the decrease in absorbance at 290 nm as described by Iwagami et al [20]. Protocatechuate 4, 5-dioxygenase (4, 5-PCDase; EC 1.13.11.8) was monitored by an increase in absorbance at 410 nm [21].…”