Characterization of a Protocatechuate Catabolic Gene Cluster from Rhodococcus opacus 1CP: Evidence for a Merged Enzyme with 4-Carboxymuconolactone-Decarboxylating and 3-Oxoadipate Enol-Lactone-Hydrolyzing Activity

摘要: The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for bacterial degradation aromatic compounds, converge at common intermediate enol-lactone. A enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than corresponding enzymes previously gamma-proteobacteria. Sequencing N terminus tryptic peptides allowed cloning gene coding enol-lactone hydrolase by using PCR with degenerate primers. showed that belongs catabolic cluster. Most interestingly, gene, usually termed pcaD, fused second pcaC, encodes enzyme catalyzing preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. two enzymatic activities could not be separated chromatographically. At least six genes catabolism appear transcribed in same direction following order: pcaH pcaG, subunits 3,4-dioxygenase, as shown N-terminal sequencing protein; pcaB due homology its product 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, PcaD PcaC activities; pcaR, presumably regulator IclR-family; designated pcaF because resembles 3-oxoadipyl coenzyme (3-oxoadipyl-CoA) thiolases. presumed pcaI, subunit succinyl-CoA:3-oxoadipate CoA-transferase, divergently pcaH.