PURIFICATION, NUCLEOTIDE-SEQUENCE AND SOME PROPERTIES OF A BIFUNCTIONAL ISOMERASE DECARBOXYLASE FROM THE HOMOPROTOCATECHUATE DEGRADATIVE PATHWAY OF ESCHERICHIA-COLI C

A 1.8-kbp region of DNA that appeared from deletion subcloning to code for 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase and 5-oxopent-3-ene -1,2,5-tricarboxylate decarboxylase was investigated further. By nucle otide sequencing, a single open reading frame was found encoding a pol ypeptide of M(r)44514. One of the deletion subclones expressed the dec arboxylase and isomerase activities at elevated levels and was used to facilitate purification of the enzyme(s). Both activities copurified, indicating that they were distinct activities of the same protein. So me kinetic properties of the purified isomerase/decarboxylase protein were investigated and it was shown that there is a 49000-fold preferen ce for 2-hydroxyhepta-2,4-diene-1,7-dioate over the structurally relat ed compound 5-carboxymethyl-2-hydroxymuconate, the substrate of a seco nd isomerase in the same catabolic pathway. Comparison of the amino ac id sequences of the two isomerases showed only a low level of similari ty, suggesting that these two enzymes are not evolutionarily related. However, comparison of the N-terminal half of the isomerase/decarboxyl ase sequence (residues 1-202) with the second half (residues 203-406) showed significant similarity, suggesting that a duplication may have occurred to produce the bifunctional gene.