soluble enzyme preparations from Escherichia coli BL21(DE3)/pSAG8(M), expressing (-)-beta-phellandrene synthase convert geranyl diphosphate to four products. beta-phellandrene (52%) as the major olefin, and lesser amounts of (1S,5S)-beta-pinene (34%), (1S,5S)-apinene (8.5%), and (4S)-limonene (6%) are identified. The stereochemistry of beta-phellandrene is not confirmed directly. Nevertheless, stereochemical considerations based on the established absolute configuration of the coproducts, and the natural occurrence of (4S)-beta-phellandrene in the turpentine, suggests that the biosynthetic product is the mechanistically related (4S)-antipode derived via the (3S)-linalyl diphosphate intermediate. The product of the ag8 gene is therefore designated (4S)-beta-phellandrene synthase
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the enzyme is extremely sensitive to thiol oxidation. Cyclase activity is completely lost when the partially purified xylem preparation is dialyzed against buffer (pH 7.8) devoid of thiol-protecting reagents (dithiothreitol or 2-mercaptoethanol). Other reducing agents, such as sodium ascorbate (5 mM) or sodium metabisulfite (5 mM), fail to protect the cyclases from inactivation, whereas readdition of 5 mM dithiothreitol to the dialyzed preparations restores 54% of the original activity