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.
The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from gamma-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.
Eulberg
D
D
Institut für Mikrobiologie, Universität Stuttgart, Germany.
Lakner
S
S
Golovleva
L A
LA
Schlömann
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics; D062385:Hydroxybenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
D062385:Hydroxybenzoates; C009091:protocatechuic acid; D002265:Carboxylic Ester Hydrolases; C021927:3-oxoadipate enol-lactonase; D002262:Carboxy-Lyases; C019832:4-carboxymuconolactone decarboxylase