<jats:title>ABSTRACT</jats:title> <jats:p> Protocatechuate 3,4-dioxygenase (EC <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="ec" xlink:href="1.13.11.3" xlink:type="simple">1.13.11.3</jats:ext-link> ) 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 <jats:italic>Streptomyces</jats:italic> sp. strain 2065 grown in <jats:italic>p</jats:italic> -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 <jats:italic>pcaGH</jats:italic> genes belonged to a 6.5-kb protocatechuate catabolic gene cluster; at least seven genes in the order <jats:italic>pcaIJFHGBL</jats:italic> appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a <jats:italic>pcaL</jats:italic> homologue which encodes a fused γ-carboxymuconolactone decarboxylase/β-ketoadipate enol-lactone hydrolase previously identified in the <jats:italic>pca</jats:italic> gene cluster from <jats:italic>Rhodococcus opacus</jats:italic> 1CP. The <jats:italic>pcaIJ</jats:italic> 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.