Formation of 4-Hydroxy-2,5-Dimethyl-3[2H]-Furanone by Zygosaccharomyces rouxii : Identification of an Intermediate

Abstract

ABSTRACT The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from d -fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium. Addition of o -phenylenediamine, a trapping reagent for α-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from d -fructose-1,6-bisphosphate under the applied growth conditions (30°C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of Z. rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of Z. rouxii cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with d -fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts.