Stereochemistry and mechanisms of the 3-carboxymuconate fungal pathway in Neurospora SY4a

Abstract

The cyclisation of cis,cis-3-carboxymuconic acid 2, catalysed by the cycloisomerase enzyme of Neurospora crassa SY4a, has been shown to occur by syn addition of the 1 -carboxy group to the 4,5-double bond to give (S)-(–)-3-carboxymuconolactone 3. Thus, the absolute configuration of the lactone 3 was determined by ozonolysis to give (S)-malic (L-malic) acid. Furthermore, incubation of trisodium cis,cis-3-carboxy-5-deuteriomuconate 9 then ozonolysis of the derived lactone 28 gave (2S,3S)-3-deuteriomalic acid 29. This evidence for syn addition was confirmed by a complementary incubation of undeuteriated 3-carboxymuconate in deuterium oxide, giving the lactone 31 and hence (2S,3R)-3-deuteriomalic acid 32.

The degradation of 3-carboxymuconolactone 3 by the multifunctional enzyme complex of Neurospora, to give 3-oxoadipic acid 5, has been studied with the deuteriated trisodium muconates 27, 14 and 20. The overall transformation has been found to involve an intramolecular, suprafacial 1,3-shift of hydrogen (or deuterium) from C-4 in the lactone to C-5 in the oxoadipic acid. The location and stereochemistry of deuterium in the oxoadipic acids 44 and 45 were established by conversion of these acids into the optically active 2-deuteriosuccinic acids 46 and 47, respectively. The 1,3-shift provides compelling evidence for the formation of the enol lactone 4 as an enzyme-bound intermediate. Successive enzymic hydrolysis and decarboxylation would then complete the biosynthesis of 3oxoadipic acid 5.