Functional Characterization of Enone Oxidoreductase Orthologs Highlights Hidden Furaneol Biosynthetic Capacity in...

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF, Furaneol) is an important flavor compound in many fruits. It is formed from the precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) by an enone oxidoreductase (EO), which has previously been characterized only in strawberry, tomato, and mango. Here, we heterologously produced EO orthologs from pineapple, kiwi, and grape, three species known to accumulate HDMF, as well as from apple and crab apple, which do not produce this compound. All candidates were expressed in fruit tissue and exhibited o-quinone oxidoreductase activity, reducing 9,10-phenanthrenequinone and various o-quinones. Importantly, all enzymes catalyzed HDMF formation, including those from apple species. The absence of HDMF accumulation in apples was attributed to the lack of the natural precursor HMMF and low levels of active EO, while HDMF was rapidly converted to its glucoside. The conserved o-quinone oxidoreductase activity across all EO orthologs suggest that fruit EOs involved in HDMF biosynthesis evolved from a broader quinone oxidoreductase family.