Formation of 5-methyl-4-hydroxy-3[2H]-furanone in cytosolic extracts obtained from Zygosaccharomyces rouxii

Formation of the flavor compound and precursor 4-hydroxy-5-methyl-3[2H]-furanone (HMF, norfuraneol) was demonstrated in cytosolic protein extracts obtained from Zygosaccharomyces rouxii after incubation with a number of carbohydrate phosphates. 4-Hydroxy-5-methyl-3[2H]-furanone was produced from D-fructose-1,6-diphosphate, D-fructose-6-phosphate, D-glucose-6-phosphate, 6-phosphogluconate, D-ribose-5-phosphate, and D-ribulose-1,5-diphosphate. Enzyme assays revealed D-fructose-1,6-diphosphatase, phosphohexose isomerase, D-glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activity in the cytosolic extracts. Model studies showed the spontaneous formation of HMF from D-ribulose-5-phosphate. It is assumed that D-ribulose-5-phosphate is generated in cytosolic extracts by the action of the investigated enzymes from the carbohydrate phosphates and is then chemically transformed to HMF. The hypothesis was proven by the production of HMF in solutions containing commercially available enzymes and [6-¹³C]-D-glucose-6-phosphate.