Abstract: Diketoreductase can catalyze a double reduction of β,δ-diketo ester to corresponding dihydroxy product with enantiomeric excess (ee) and diastereomeric excess (de) both greater than 99.5%.In order to explore the catalytic mechanism of this unique enzyme,the present study investigated the diketone reduction process and reaction characteristics by diketoreductase.We found that two mono-hydroxy intermediates were produced during the diketone reduction and its reverse reaction.The two intermediates were further separated by C₁₈column chromatography and structurally confirmed by chiral HPLC with authentic standards.Two mono-hydroxy intermediates could be served as the substrates for the reduction by diketoreductase with different reaction velocities.The formation and disappearance of intermediates were largely affected by temperature and substrate concentration.In addition,steady state kinetics with the two intermediates showed different reaction rates in their disappearance.Collectively,the results indicate that the diketone reduction undergoes a two-step process with the formation of both intermediates,but the disappearance rates for the two intermediates are slightly different.