Phenylbutanone raspberry ketone, accumulating in the mature fruits of raspberry (Rufous idaeus), imparts the characteristic aroma to the fruits. Here we describe the isolation and characterization of raspberry ketone/zingerone synthase 1 (RZS1), which catalyzed the NADPH-dependent reduction of 4-hydroxybenzalacetone and 3-methoxy-4-hydroxybenzalacetone to raspberry ketone and zingerone (the latter not found in raspberry), respectively. Its apparent K-m values for 4-hydroxybenzalacetone and NADPH were 88 mu M and 202 mu M, respectively. RZS1 preferred 4-hydroxybenzalacetone to 3-methoxy-4-hydroxybenzalacetone as a substrate by a factor of 1.7, and showed a 6-fold preference for 4-hydroxybenzalacetone over p-coumaraldehyde, and no activity for coniferaldehyde. Expression analysis of the RZS1 gene throughout the plant revealed that its transcript level was highest in mature fruits. We conclude that RZS1 is responsible for hydrogenation of the alpha,beta-unsaturated double bond of phenylbutenones, the final step of the raspberry ketone biosynthesis, in the raspberry fruits. (C) 2011 Elsevier Inc. All rights reserved.