The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: Enzymology of 3-methylmuconolactone metabolism

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a mo dified beta-ketoadipate pathway. This organism has been shown to activate 3 -methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysi s of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chrom atography, gel filtration, and anion-exchange chromatography. The enzyme, t ermed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a nati ve M-r of 128,000, and a subunit M-r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone s ubstrate and displays little or no activity with other monoene and diene la ctone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA syn thetase reaction with its natural substrate.