Leukotriene B412-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB412-HD/PGR) responsible for the reduction of a double-bond of theα,β-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670

CS-670 is a non-steroidal anti-inflammatory agent with an alpha,beta-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1'R,2'S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the alpha,beta-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B(4) 12-hydroxydehydrogenase/15-ketoprostaglandinDelta13-reductase (LTB(4) 12-HD/PGR). The identification was confirmed by cloning LTB(4) 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF(2alpha), benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB(4) 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.