Properties of fungal 3-carboxymuconate cyclases

3-Carboxy-muconate cyclase [3-carboxymuconolactone lyase (decyclizing) EC.5.5.1.5] is a unique fungal enzyme involved in the dissimilation of protocatechuate via 3-oxoadipate. This enzyme mediates the lactonization of 3-carboxy-cis,cismuconate to 3-carboxymuconolactone whereas in bacteria the same intermediate is lactonized to 4-carboxymuconolactone, by 3-carboxymuconate cycloisomerase [4-carboxymuconolactone lyase (decyclizing) EC.5.5.1.2]. The results of the detailed investigations on the bacterial enzymes of the 3-oxoadipate pathway have been the subject of extensive evolutionary discussions. The fungal 3-carboxymuconate lactonizing enzyme, which determines the divergence between the procaryotic and eucaryotic pathways has therefore raised a considerable interest. The Aspergillus niger L6 cyclase has been previously purified and some of its properties investigated. It has also been used to raise antiserum which cross-reacted with extracts of several fungi imperfectii, induced by growth on p-hydroxybenzoate or protocatechuate, but not with the analogous lactonizing enzyme from Pseudomonas putida. In the present work an improved purification method of the A. niger L6 cyclase, involving ammonium sulphate fractionation, anion-exchange chromatography, gel filtration, hydrophobic chromatography and chromatofocusing achieved a 3380-fold purification of the enzyme. The native molecule behaved as a molecule with an apparent mol.wt. of 162 000 by calibrated gel permeation chromatography and its subunit size was 42 000 ± 1 000 by SDS-polyacrylamide gel electrophoresis. The enzyme appears therefore to be tetrameric. Its Michaelis constant at the optimum pH is 15pM. The product, 3-carboxymuconolactone, the substrate analogue 1-methyl 3-carboxy-cis,cis-muconate and several metallic ions are inhibitory to the enzyme. In the concentration range tested, cis,cis-muconate and the other isomeric muconic acids did not affect the enzyme activity. The isoelectric point determined by chromatofocusing is 4.85 ± 0.05. Other fungal cyclases which are immunologically related to the A. niger enzyme appear also to be tetrameric, with subunit sizes in the range 38 500-43 000 and molecular sizes of 150 000-180 000, except the Scopulariopsis Candida cyclase which behaved as a larger molecule by calibrated gel permeation chromatography. The Michaelis constants of these enzymes fall in a narrow range (12-17(µM) but their isoelectric points vary between 4.2 and 6.0. Possible evolutionary implications of these results are discussed.