Absolute stereochemical course of the 3-carboxymuconate cycloisomerases from Pseudomonas putida and Acinetobacter colcoaceticus: Analysis and implications

Article abstract of DOI:10.1021/ja00252a034

The absolute stereochemical course of the 3-carboxymuconate cycloisomerases [EC 5.5.1.2; 2-carboxy-5-oxo-2,5-dihydrofuran-2-acetate lyase (decyclizing)] from Pseudomonas putida and Acinetobacter calcoaceticus has been determined by chemical and ¹H NMR methods. The product of the enzyme-catalyzed reaction in ²H₂O was detected by NMR and trapped by catalytic hydrogenation to afford 5-[²H]homocitrate lactone. Subsequent chemical degradation of the monodeuteriated homocitrate lactone gave (2r,3S)-2-[²H]citrate as determined by ¹H NMR analysis. The product of the cycloisomerase reaction was established as (4R,5R)-5-[²H]-4-carboxymuconate, indicating that the lactonization proceeded by an anti addition - the mechanistic and stereochemical antipode of the previously studied muconate cycloisomerase from P. putida and 3-carboxymuconate cycloisomerase from Neurospora crassa. The anti addition probably represents the lower energy pathway for the reaction and suggests that the evolutionary relationship between the two classes of cycloisomerases is more remote than previously believed.