Cloning and complete nucleotide sequence determination of the catB gene encoding m,m-muconate lactonizing enzyme

Abstract

The enzyme, cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1), has been proposed to play a key role in the β-ketoadipate pathway of benzoate degradation. A 10.2-kb EcoRI fragment isolated from a Pseudomonas putida genomic library complemented a mutant deficient in this enzyme. The MLEI coding gene, catB, was localized to a 1.6-kb fragment which was sequenced by the dideoxy chain termination method. MLEI was purified 25-fold from crude extracts of benzoate-grown P. putida PRS2015 harboring the cloned catB gene. Purified MLEI was greater than 95 % homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit Mr was 40000 which was in close agreement with the nucleotide sequence data. N-terminal sequence analysis of purified MLEI protein agreed with the N terminus predicted by the nucleotide sequence. Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene (K.L. Ngai B.F., D.K. Chatterjee L.N. Ornston, and A.M.C., unpublished), whose gene product catalyzes the analogous reaction in 3-chlorobenzoate degradation, showed significant homology. These results suggest that catB and clcB have diverged from a common ancestral gene.