Substrate specificity of α-humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase alpha-humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E-GPP) and neryl pyrophosphate (2Z-NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E-FPP). Also, gas chromatography mass spectrometry (GS-MS) was carried out to identify the products of the bioconversion of (2E)-GPP and (2Z)-NPP.