A 2,585 bp chromosomal DNA segment of Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) which contains a gene cluster encoding part of the modified ortho-cleavage pathway encodes a putative transport protein for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase and methylmuconolactone isomerase. The putative 4-methylmuconolactone transporter, a protein with a calculated molecular mass of 45.8 kDa, exhibits sequence homology to other members of the major superfamily of transmembrane facilitators and shows the common structural motif of 12 transmembrane-spanning alpha-helical segments and the hallmark amino acid motif characteristic of the superfamily. Consistent with the novelty of the reaction catalyzed by 4-methylmuconolactone methylisomerase, no primary sequence homologies were found between this enzyme or its gene and other proteins or genes in the data banks, suggesting that this enzyme represents a new type of isomerase. The molecular mass of the native 4-methylmuconolactone methylisomerase was determined by gel filtration analysis to be 25 +/- 2 kDa. From the polynucleotide sequence of the gene, a molecular mass of 12.9 kDa was calculated and hence we predict a homodimeric quaternary structure. The high sensitivity of 4-methylmuconolactone methylisomerase to heavy metals and thiol-modifying reagents implicates the involvement of sulfhydryl groups in the catalytic reaction. The methylmuconolactone isomerase - calculated molecular mass 10.3 kDa - has a primary structure related to the classical muconolactone isomerases (EC 5.3.3.4) of Acinetobacter calcoaceticus, of two Pseudomonas putida strains and of Ralstonia eutropha JMP134, suggesting that these are all isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity.