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

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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 β-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 β- and α-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 γ-carboxymuconolactone decarboxylase/β-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.