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 nucleotide sequencing, a single open reading frame was found encoding a polypeptide of M(r)44514. One of the deletion subclones expressed the decarboxylase 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. Some kinetic properties of the purified isomerase/decarboxylase protein were investigated and it was shown that there is a 49,000-fold preference for 2-hydroxyhepta-2,4-diene-1,7-dioate over the structurally related compound 5-carboxymethyl-2-hydroxymuconate, the substrate of a second isomerase in the same catabolic pathway. Comparison of the amino acid sequences of the two isomerases showed only a low level of similarity, suggesting that these two enzymes are not evolutionarily related. However, comparison of the N-terminal half of the isomerase/decarboxylase 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.
Roper
D I
DI
Department of Biochemistry, University of Leicester, England.
Cooper
R A
RA
D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D015102:3,4-Dihydroxyphenylacetic Acid; D002262:Carboxy-Lyases; C031940:5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase; D007535:Isomerases; C084023:2-hydroxyhepta-2,4-diene-1,7-dioate isomerase; D019748:Carbon-Carbon Double Bond Isomerases