Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134.
4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.
Pieper
D H
DH
Institut für Mikrobiologie, Universität Stuttgart, Federal Republic of Germany.
Stadler-Fritzsche
K
K
Knackmuss
H J
HJ
Engesser
K H
KH
Bruce
N C
NC
Cain
R B
RB
D000272:Adipates / Q000378:metabolism*; D000421:Alcaligenes / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids; D011549:Pseudomonas / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D000272:Adipates; C027316:3-oxoadipic acid; D007535:Isomerases; D019751:Intramolecular Transferases; C054260:4-carboxymethyl-methylbut-2-ene-1,4-olide isomerase