A single amino acid determines the site of deprotonation in the active center of sesquiterpene synthases SbTPS1 and SbTPS2 from Sorghum bicolor.

The multitude of terpene carbon skeletons found in nature is formed by enzymes known as terpene synthases (TPSs). These proteins are often multiproduct enzymes converting a single prenyl diphosphate substrate into a mixture of terpene products. The recently identified sesquiterpene synthases SbTPS1 and SbTPS2 from Sorghum bicolor produce terpene blends containing the same products, but in different proportions. A single amino acid in the active site was reported to determine the different product specificities of SbTPS1 and SbTPS2. In this study we examined the reaction mechanism of the Sorghum TPSs. Feeding experiments with deuterium-labeled substrates and chiral analysis of the enzyme products zingiberene, β-sesquiphellandrene and β-bisabolene revealed that the reactions catalyzed by both enzymes proceeded via (S)-nerolidyl diphosphate and the cyclic (6S)-bisabol-7-yl and (6R)-bisabol-1-yl cation intermediates. The site of deprotonation of the final cation was shown to be the only catalytic difference between SbTPS1 and SbTPS2. Docking of the (6R)-bisabol-1-yl cation into structural models of SbTPS1 and SbTPS2 indicated a potential role of initially cleaved pyrophosphate group as a proton acceptor.