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

Abstract

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis 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 chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

FIG. 1

Effect of CoA and ATP on the degradation of 3-methylmuconolactone. 3-Methylmuconolactone was incubated with dialyzed crude extract from benzoate-grown cells (□), dialyzed crude extract from p-toluate-grown cells in the absence of CoA and ATP (▵), and dialyzed crude extract in the presence CoA and ATP (○). 3-Methylmuconolactone degradation was monitored by HPLC.

FIG. 2

Formation of a β-ketoacyl-CoA thioester. The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.0), 0.2 mM (○) or 0.5 mM (•) 3-methylmuconolactone, 0.5 mM CoA, 1 mM ATP, 1 mM MgCl₂, and 18 mU of 3-methylmuconolactone-CoA synthetase in a total volume of 1 ml. After a 30-min incubation, crude extract from p-toluate-grown cells (0.26 mg of protein) or benzoate-grown cells (0.21 mg of protein [□]) was added to the reaction mixture and the reaction was monitored by determining A₃₀₅. ▵, control with no crude extract.

FIG. 3

Proposed pathway for the dissimilation of 3-methylmuconolactone in Rhodococcus rhodochrous N75.